FLUORESCENT pH DETECTOR SYSTEM AND RELATED METHODS

ABSTRACT

Fluorescent pH detector and methods for measuring pH using the fluorescent pH detector.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/491,063, filed May 27, 2011, and U.S. Provisional Application No.61/650,304, filed May 22, 2012, each expressly incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Optical sensors (optrodes) for measuring pH are well known. Certainaromatic organic compounds (like phenolphthalein) change color with pHand can be immobilized on solid supports to form “pH paper.” Thesevisual indicators are easy to use, but do not provide a quantitativereading. The color changes can be difficult to distinguish accurately,and can be masked by colored analyte. Fluorescent indicators have alsobeen used as optical sensors. pH Sensitive fluorescent dyes can beimmobilized on solid supports and generally are more sensitive incomparison to the simple color changing (absorbance or reflectancebased) indicators. The improved sensitivity of fluorescent indicatorsallows the solid support to be miniaturized, and this has been used toadvantage in development of fiber optic sensor devices for measuring pH,CO₂, and O₂ parameters in blood.

A specific need in the medical industry exists for accurate pHmeasurement of blood. The pH of blood, or other bodily fluids (pleuraleffusions) can be associated with certain physiologic responsesassociated with pathology. Blood gas analyzers are common critical careinstruments. Depending on storage conditions, the pH of separated bloodcomponents (plasma, platelets) can change rapidly due to off-gassing ofdissolved CO₂ from the enriched venous blood that is collected from adonor. Platelets in particular are metabolically active, and generatelactic acid during storage at 20-22° C. European quality guidelines forplatelets prepared by the “buffycoat method” require pH of storedplatelets to be pH 6.8-7.4 at 37° C. (7.0-7.6 at 22° C.).

Seminaphthofluorescein (SNAFL) compounds and the relatedseminaphthorhodafluor (SNARF) compounds are commercially availableratiometric fluors (Molecular Probes, Inc., Eugene, Oreg.; see, forexample, U.S. Pat. No. 4,945,171) and their synthesis and spectralproperties have been described. These compounds have advantagesincluding long wavelength absorbance that can be efficiently excitedwith LED light sources. Relevant acid/base equilibria and associatedspectral properties are shown below.

Deprotonation of the naphthol structure of SNAFL dyes gives anaphtholate molecule with longer wavelength fluorescence emission. ThepKa is the pH value where the two molecular species form in equalamounts. SNAFL compounds with reactive linker groups that allow theirconjugation to other molecules of interest are also commerciallyavailable.

Various methods have been used to immobilize “ratiometric” dyes to solidsupports for use in fiber optic pH detectors. Carboxynaphthofluorescein(CNF) has been conjugated to aminoethyl-cellulose and this material wasglued to polyester (Mylar) films to make sensing membranes for optrodes.The pKa of this material was 7.41, slightly lower than the free CNF (pKa7.62). The use of tetraethoxysilane to trap CNF in a sol-gel glass thatwas formed on glass cover slips has also been reported. The pKa of thismaterial was 7.46. A 9-chloro substituted SNAFL analog (SNAFL-2) hasbeen reacted with polyvinylamine and the residual amino groups werecrosslinked with a photocrosslinker to form a gel-like coating onacrylic fibers. The pKa of this fiber-optic sensor was 7.14,significantly lower than the published pKa of the free SNAFL compound(pKa ˜7.7). This shows that molecular environment and linker structuresurrounding the immobilized dye can alter the performance of a pHdetector.

Despite the advances made in the detection of pH noted above, thereexists a need for improved methods and devices for measuring pH. Thepresent invention seeks to fulfill this need and provides furtherrelated advantages.

SUMMARY OF THE INVENTION

The present invention provides methods and systems for measuring the pHof a sample using the fluorescent pH detector.

In one aspect, the invention provides a method for measuring the pH of asample. In one embodiment, the method comprises:

(a) irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe,

wherein the fluorescent species immobilized on the substrate is inliquid communication with a sample,

wherein the excitation light has a wavelength sufficient to effectfluorescent emission from the fluorescent species,

wherein the fluorescent species exhibits a first emission intensity at afirst emission wavelength and a second emission intensity at a secondemission wavelength, the ratio of the first and second emissionintensities being dependent on pH, and

wherein the fluorescent species immobilized on the substrate is preparedby covalently coupling a benzo[c]xanthene linker to the substrate,wherein the benzo[c]xanthene linker compound has formulae (I) or (II)

their salts, active esters, acid/base forms, and tautomers, wherein

at least one of the X₀ groups is a halogen (e.g., chloro);

R_(1′), R₁, R₂, R₃, and R₄ are each independently selected fromhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl,(C₁-C₈)alkylthio and (C₁-C₈)alkoxy, aryl, and heteroaryl; X₁, X₂, X₃,and X₄ are each independently selected from the group consisting ofhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl,aryl(C₁-C₄)alkyl, heteroaryl, SO₃H, and CO₂H; wherein the alkyl portionsof any of R_(1′), R₁, R₂, R₃, and R₄, and X₁, X₂, X₃, and X₄ areoptionally substituted with halogen, carboxy, sulfo, amino, mono- ordialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and whereinthe aryl or heteroaryl portions of any of R_(1′), R₁, R₂, R₃, and R₄,and X₁, X₂, X₃, and X₄ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; and

wherein the linker arm has the formula

—CH₂CH₂-L₁-NH-L₂-FG

wherein L₁ has a length not exceeding the length of a normal alkyl chainof 25 carbons and comprises from one to about 50 atoms,

wherein L₂ has a length not exceeding the length of a normal alkyl chainof 25 carbons and comprises from one to about 50 atoms, and

wherein FG is a functional group reactive toward and capable ofcovalently coupling the fluorescent dye compound to a suitably reactivematerial; and

(b) measuring the first and second emission intensities to determinedthe pH of the sample.

In one embodiment, the benzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.

In one embodiment, the benzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.

Suitable substrates include macromolecules such as albumins. In oneembodiment, the macromolecule is a human serum albumin.

In certain embodiments, the sample is blood or a blood product. In oneembodiment, the sample is contained within a sealed vessel.

In one embodiment, the probe is physically isolated from the fluorescentspecies immobilized on the substrate by a window transparent to theexcitation light and the fluorescent emission.

In another aspect of the invention, a system for measuring pH isprovided. In one embodiment, the system comprises:

(a) a light source for exciting a fluorescent species immobilized on thesubstrate, wherein the fluorescent species has a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength,

wherein the fluorescent species immobilized on the substrate is preparedby covalently coupling a benzo[c]xanthene linker to the substrate,wherein the benzo[c]xanthene linker compound has formulae (I) or (II)

their salts, active esters, acid/base forms, and tautomers, wherein

at least one of the X₀ groups is a halogen (e.g., chloro).

R_(1′), R₁, R₂, R₃, and R₄ are each independently selected fromhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl,(C₁-C₈)alkylthio and (C₁-C₈)alkoxy, aryl, and heteroaryl; X₁, X₂, X₃,and X₄ are each independently selected from the group consisting ofhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl,aryl(C₁-C₄)alkyl, heteroaryl, SO₃H, and CO₂H; wherein the alkyl portionsof any of R_(1′), R₁, R₂, R₃, and R₄, and X₁, X₂, X₃, and X₄ areoptionally substituted with halogen, carboxy, sulfo, amino, mono- ordialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and whereinthe aryl or heteroaryl portions of any of R_(1′), R₁, R₂, R₃, and R₄,and X₁, X₂, X₃, and X₄ are optionally substituted with from one to foursubstituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; and

wherein the linker arm has the formula

—CH₂CH₂-L₁-NH-L₂-FG

wherein L₁ has a length not exceeding the length of a normal alkyl chainof 25 carbons and comprises from one to about 50 atoms,

wherein L₂ has a length not exceeding the length of a normal alkyl chainof 25 carbons and comprises from one to about 50 atoms, and

wherein FG is a functional group reactive toward and capable ofcovalently coupling the fluorescent dye compound to a suitably reactivematerial;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(g) a probe housing the distal termini of the excitation lightguide,first emission lightguide, and second emission light guide; and

(h) an assembly for receiving the probe, the assembly comprising:

(i) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light,

(ii) a tip member reversibly connectable to the housing's second end,wherein the tip member is adapted to receive liquid from a sample to bemeasured, and

(iii) the fluorescent species immobilized on the substrate intermediatethe tip member and the window, wherein the fluorescent speciesimmobilized on the substrate is in liquid communication with the sampleduring the measurement.

In one embodiment, the benzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.

In one embodiment, the benzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.

Suitable substrates include macromolecules such as albumins. In oneembodiment, the macromolecule is a human serum albumin.

In one embodiment, the probe is physically isolated from the fluorescentspecies immobilized on the substrate by a window transparent to theexcitation light and the fluorescent emission.

In one embodiment, the light source is a light-emitting diode.

In one embodiment, the first and second detectors are photodiodes.

In one embodiment, the sample is blood or a blood product. In oneembodiment, the sample is contained within a sealed vessel.

In a further aspect, the invention provides a method for measuring thepH of a sample. In one embodiment, the method comprises:

(a) irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe,

wherein the fluorescent species immobilized on the substrate is inliquid communication with a sample,

wherein the excitation light has a wavelength sufficient to effectfluorescent emission from the fluorescent species,

wherein the fluorescent species exhibits a first emission intensity at afirst emission wavelength and a second emission intensity at a secondemission wavelength, the ratio of the first and second emissionintensities being dependent on pH, and

wherein the fluorescent species immobilized on the substrate is preparedby covalently coupling a benzo[c]xanthene linker to the substrate,wherein the benzo[c]xanthene linker compound has formula (III)

its salts, active esters, acid/base forms, and tautomers, wherein

at least one of the X₀ groups is a halogen (e.g., chloro).

R_(1′), R_(1″), R₁, R₂, R₃, and R₄ are each independently selected fromhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl,(C₁-C₈)alkylthio and (C₁-C₈)alkoxy, aryl, and heteroaryl; X₁, X₂, and X₃are each independently selected from the group consisting of hydrogen,halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl,aryl(C₁-C₄)alkyl, heteroaryl, SO₃H, and CO₂H; wherein the alkyl portionsof any of R_(1′), R_(1″), R₁, R₂, R₃, and R₄, and X₁, X₂, and X₃ areoptionally substituted with halogen, carboxy, sulfo, amino, mono- ordialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and whereinthe aryl or heteroaryl portions of any of R_(1′), R_(1″), R₁, R₂, R₃,and R₄, and X₁, X₂, and X₃ are optionally substituted with from one tofour substituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; and

wherein the linker arm is a carboxylic acid group (i.e., —CO₂H); and

(b) measuring the first and second emission intensities to determinedthe pH of the sample.

In another aspect of the invention, a system for measuring pH isprovided. In one embodiment, the system comprises:

(a) a light source for exciting a fluorescent species immobilized on thesubstrate, wherein the fluorescent species has a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength,

wherein the fluorescent species immobilized on the substrate is preparedby covalently coupling a benzo[c]xanthene linker to the substrate,wherein the benzo[c]xanthene linker compound has formula (III)

its salts, active esters, acid/base forms, and tautomers, wherein

at least one of the X₀ groups is a halogen (e.g., chloro).

R_(1′), R_(1″), R₁, R₂, R₃, and R₄ are each independently selected fromhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl,(C₁-C₈)alkylthio and (C₁-C₈)alkoxy, aryl, and heteroaryl; X₁, X₂, and X₃are each independently selected from the group consisting of hydrogen,halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl,aryl(C₁-C₄)alkyl, heteroaryl, SO₃H, and CO₂H; wherein the alkyl portionsof any of R_(1′), R_(1″), R₁, R₂, R₃, and R₄, and X₁, X₂, and X₃ areoptionally substituted with halogen, carboxy, sulfo, amino, mono- ordialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and whereinthe aryl or heteroaryl portions of any of R_(1′), R_(1″), R₁, R₂, R₃,and R₄, and X₁, X₂, and X₃ are optionally substituted with from one tofour substituents selected from the group consisting of halogen, cyano,carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy; and

wherein the linker arm is a carboxylic acid group (i.e., —CO₂H);

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(g) a probe housing the distal termini of the excitation lightguide,first emission lightguide, and second emission light guide; and

(h) an assembly for receiving the probe, the assembly comprising:

(i) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light,

(ii) a tip member reversibly connectable to the housing's second end,wherein the tip member is adapted to receive liquid from a sample to bemeasured, and

(iii) the fluorescent species immobilized on the substrate intermediatethe tip member and the window, wherein the fluorescent speciesimmobilized on the substrate is in liquid communication with the sampleduring the measurement.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1A is a schematic illustration of a representative system of theinvention for measuring pH;

FIG. 1B is a schematic illustration of an optical platform useful in thesystem of the invention for measuring pH;

FIG. 2 is a schematic illustration of a representative housing forexcitation and emission light guides useful in the system of theinvention;

FIG. 3 illustrates the relationship between the excitation/emissionoptical fiber housing and the sealed vessel port;

FIGS. 4A-4C illustrate a representative port assembly for introducing asubstrate-immobilized fluorescent species into a sealed vessel, FIG. 4Aillustrates the assembled port, FIG. 4B is an exploded view of the portassembly; and FIG. 4C is a plan view of tip;

FIGS. 5A and 5B illustrate representative sealed vessels incorporatingthe substrate-immobilized fluorescent species, FIG. 5A shows a sealedvessel in which substrate was introduced through process of puncture andreseal, FIG. 5B shows a sealed vessel incorporating substrate duringvessel manufacture;

FIG. 6 is a representative port assembly useful in the manufacture of asealed vessel;

FIGS. 7A-E illustrate the structures of representativeseminaphthofluorescein compounds useful in the method and system of theinvention;

FIG. 8 illustrates the emission spectra as a function of pH of arepresentative fluorescent species (SNAFL-1) useful in the method andsystem of the invention;

FIG. 9 illustrates the emission spectra as a function of pH of arepresentative fluorescent species (EBIO-3) useful in the method andsystem of the invention;

FIG. 10 is a schematic illustration of the preparation of arepresentative fluorophore-protein (EBIO-3/HSA) conjugate useful in themethod and system of the invention;

FIG. 11 illustrates the emission spectra as a function of pH of arepresentative fluorophore-protein conjugate (SNAFL-1/HSA) useful in themethod and system of the invention;

FIG. 12 illustrates the emission spectra as a function of pH of arepresentative fluorophore-protein conjugate (EBIO-3/HSA) useful in themethod and system of the invention;

FIG. 13 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (SNAFL-1/HSA) as afunction of pH (Oxyphen);

FIG. 14 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (SNAFL-1/HSA) as afunction of pH (nitrocellulose);

FIG. 15 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (EBIO-3/HSA) as afunction of pH (nitrocellulose);

FIG. 16 illustrates the data used in the method of the invention formeasuring pH;

FIG. 17 illustrates the results of the method of the invention forplatelet rich plasma;

FIG. 18 illustrates the correlation of pH results for platelet richplasma obtained by the method and system of the invention;

FIG. 19 illustrates stability of a representative substrate-immobilizedfluorophore conjugate of the invention;

FIG. 20 illustrates a representative device of the invention formeasuring carbon dioxide in a sealed vessel;

FIG. 21 illustrates the effect of probe position on fluorescentintensity in measuring pH in accordance with the invention;

FIG. 22 illustrates the effect of membrane pore size on fluorescentintensity in measuring pH in accordance with the invention;

FIG. 23 illustrates the synthesis of a representative 2-haloseminaphthofluorescein compound (BCSI-3) useful in the method and systemof the invention;

FIG. 24 compares the absorbance spectra of two representativefluorescent species (EBIO-3 and BCSI-3) at pH 9.5 (borate buffer);

FIG. 25 compares the absorbance spectra of two representativefluorescent species (EBIO-3 and BCSI-3) at pH 7.4 (phosphate bufferedsaline);

FIG. 26 compares the absorbance spectra of two representativefluorescent species (EBIO-3 and BCSI-3) at pH 4.5 (acetate buffer);

FIG. 27 illustrates the pKa determination of a representativefluorescent species (EBIO-3) by plotting peak absorbance as a functionof pH;

FIG. 28 illustrates the pKa determination of a representativefluorescent species (BCSI-3) by plotting peak absorbance as a functionof pH;

FIG. 29 compares the photodegradation of two representative fluorescentspecies (EBIO-3 and BCSI-3) at pH 7.4 (phosphate buffered saline) byplotting percent reduction in absorbance at 530 nm as a function ofexposure time to light;

FIG. 30 is a schematic illustration comparing the preparation of tworepresentative fluorophore-protein conjugates (EBIO-3/HSA andBCSI-3/HSA) useful in the method and system of the invention;

FIG. 31 is a graph illustrating loading ratio of a representativefluorescent species (BCSI-3) to a representative protein (rHSA) as afunction of offering ratio (BCSI-3/rHSA); and

FIG. 32 compares fluorescent ratio signal as a function of test bufferpH for representative fluorophore-protein conjugates (EBIO-3/HSA andBCSI-3/HSA conjugates) useful in the method and system of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for measuring pH and a systemfor measuring pH. The method and system are suited to measure the pH ofa specimen contained in a sealed container.

In one aspect of the invention, a method for measuring pH is provided.In the method, pH is determined by comparing fluorescent emissionintensities from a single fluorescent species having pH-dependentfluorescent emission. The fluorescent species having pH-dependentfluorescent emission has a first emission intensity at a firstwavelength and a second emission intensity at a second wavelength, thefirst and second emission intensities being characteristic of pH in theenvironment of the fluorescent species. The ratio of the first andsecond emission intensities provides pH measurement. Calibration of thefirst and second emission intensities provides an intensity-basedreference (ratio information) that is used to determine the pH of theenvironment of the fluorescent species.

The method of the invention is a fluorescent wavelength-ratiometricmethod. As used herein, the term “fluorescent wavelength-ratiometric”refers to the method by which the first and second fluorescent emissionintensities measured at first and second emission wavelengths,respectively, are ratioed to provide pH information.

In the method, the fluorescent species having pH-dependent fluorescentemission is immobilized on a substrate in contact with the sample suchthat the fluorescent species is in contact with the sample. Theimmobilized fluorescent species in contact with the sample is located inthe sample such that the fluorescent species can be interrogated. Thefluorescence measurement is made by irradiating the fluorescent speciesat a wavelength sufficient to elicit fluorescent emission, which is thenmeasured. Because of the pH-dependent nature of the fluorescent species'emission profile (i.e., first and second fluorescent emissionintensities measured at first and second emission wavelengths,respectively) the measurement of the fluorescent emission profile yieldsthe pH of the fluorescent species' environment (i.e., sample pH).

In one embodiment of the method of the invention, the sample for whichthe pH is to be determined is contained in a sealed vessel. This methodis suitable for measuring pH of blood and blood products sealed in aconventional blood storage vessel.

In another embodiment, the sample for which the pH is to be determinedis contained in an open vessel. As used herein, the term “open vessel”refers to a vessel that is not sealed. This method is suitable wherecontamination of the sample being measured is to be avoided. In thismethod, the probe is cleaned and/or sterilized and is used once anddiscarded. This method is suitable for measuring the pH of materialsused in food, pharmaceutical, or biological research where the vesselcontaining the material is not sealed (i.e., open). Such a “lab-use”system includes a tip (see description below) placed onto the probe. ThepH measurement is made by immersing the tip into the sample andmeasuring pH. The tip is removed from the sample, removed from theprobe, and discarded.

In the method for measuring the pH of a contained sample, thesubstrate-immobilized fluorescent species is introduced into the vesseleither before or after the sample is placed in the vessel. As usedherein, the term “sealed vessel” refers to a vessel that prevents itscontents from exposure to the environment exterior to the vessel. Thesealed vessel prevents the contents of the vessel from contact from, forexample, liquids and gases outside of the vessel. The sealed vessel alsoprevents the contents of the vessel from escaping the vessel.

The vessel can be manufactured to include the substrate-immobilizedfluorescent species as a component of the vessel. In such an embodiment,the substrate-immobilized fluorescent species is incorporated into thevessel during manufacture to provide a vessel into which a sample can belater introduced and its pH measured. The manufacture of a vesselincorporating the substrate-immobilized fluorescent species is describedin Example 1.

Alternatively, the substrate-immobilized fluorescent species can beintroduced into the vessel after the sample has been introduced into thevessel. In such an embodiment, the substrate-immobilized fluorescentspecies is introduced into the vessel by a process in which the vesselis first punctured (or spiked) to introduce the substrate-immobilizedfluorescent species and then resealed to provide a sealed vesselincluding the sample now in contact with the substrate-immobilizedfluorescent species. The process for introducing thesubstrate-immobilized fluorescent species into a sealed vessel isdescribed in Example 2.

As noted above, the vessel including the substrate-immobilizedfluorescent species in contact with the sample is sealed before, during,and after interrogation. Interrogation of the fluorescent speciesrequires excitation of the species at a wavelength sufficient to effectfluorescent emission from the species and measurement of thatfluorescent emission. In the method of the invention, interrogation isaccomplished through a window in the sealed vessel. The fluorescentspecies is excited by irradiation through the window, and emission fromthe fluorescent species is collected from the fluorescent species thoughthe window. The window is a component of the sealed vessel and allowsfor interrogation of the fluorescent species in contact with the sample.The window is sufficiently transparent at the excitation and emissionwavelengths to permit interrogation by the method. Thesubstrate-immobilized fluorescent species is positioned in proximity tothe window sufficient for interrogation: proximity sufficient toeffectively excite the fluorescent species and to effectively collectemission from the fluorescent species. It will be appreciated that forepifluorescence applications, a single window is used. However, othermethods and devices of the invention can include other optical paths,such as straight-through or right angle optical paths, where more thanone window can be used.

The method of the invention includes irradiating thesubstrate-immobilized fluorescent species, which in one embodiment iscontained along with a sample in a sealed vessel, at a wavelengthsufficient to effect emission from the fluorescent species and tomeasure that emission. Exciting light and fluorescent emission passthrough the sealed vessel's window. In one embodiment, the sealed vesselfurther includes a port for receiving a housing that holds theexcitation light guide and emission light guide. In one embodiment, theexcitation light guide includes one or more optical fibers that transmitthe excitation light from a light source to the fluorescent species. Inone embodiment, the emission light guide includes one or more opticalfibers that transmit the emission light from the fluorescent species toa light detector. The port receiving the housing is positioned inproximity to the window sufficient for interrogation: proximitysufficient to effectively excite the fluorescent species and toeffectively collect emission from the fluorescent species.

As with all optical fluorescent methods, the method of the inventionincludes a light source for exciting the fluorescent species and adetector for measuring the emission of the fluorescent species. Lightsources, wavelength selection filters, and detectors are selected basedon the absorbance and emission profiles of the fluorescent species usedin the method.

Suitable light sources provide excitation energy at a wavelength andintensity sufficient to effect fluorescent emission from the fluorescentspecies. The light source can provide relatively broad wavelength bandexcitation (e.g., ultraviolet or white light sources) or relativelynarrower wavelength band excitation (e.g., laser or light-emittingdiode). To enhance excitation efficiency and emission measurement,relatively broad wavelength band exciting light from the source can beselected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitablelight sources include tungsten lamps, halogen lamps, xenon lamps, arclamps, LEDs, hollow cathode lamps, and lasers.

Suitable detectors detect the intensity of fluorescent emission over theemission wavelength band of the fluorescent species. To enhance emissionmeasurement, fluorescent emission from the fluorescent species sourcecan be selected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitabledetectors include photomultiplier tubes and solid state detectors, suchas photodiodes, responsive to the wavelength emission band of thefluorescent species. Other suitable detectors are photovoltaic cells,PIN diodes, and avalanche photodiodes.

Through the use of filters, all of the excitation light that reflectsoff the target is filtered out before reaching the detector. This can beachieved by using filters in both the excitation and emission opticalpaths. In certain instances, reflected excitation light (which is manyorders of magnitude more intense than the emission light) that reachesthe detector can swamp the specific signal. Generally, 10E5 (10⁵) orgreater out-of-band rejection is appropriate in each of the filter sets.Reduction of excitation light can also be achieved by using an angledwindow so that reflected light is directed away from the emissiondetector. However, such an optical path is not as effective as filtersets.

Excitation light from the source can be directed to the fluorescentspecies through the use of a light guide, such as one or more opticalfibers. Similarly, emission from the fluorescent species can be directedto the detector through the use of a light guide, such as one or moreoptical fibers.

A representative system for carrying out the method of the invention isillustrated schematically in FIG. 1A. Referring to FIG. 1A, system 100includes controller 110 that controls and operates the systemcomponents. System components include keypad 120 for inputtinginformation including system commands; display 130 for determining thestatus of the system and viewing pH determination results; barcodereader 140 for inputting information to the system including theidentification of the sample, the pH of which is to be measured by thesystem; printer 150 for printing system status and pH determinationresults; battery (or wall plug and power adapter) 160 for powering thesystem; memory device 165 for storing test results and calibration data;signal processing electronics 170 for commanding the optical platformcomponents and processing signals from the optical platform; and opticalplatform 180 including an excitation source, emission detectors, lightguides, and associated lenses and filters. Optical platform includesprobe member 185 housing one or more excitation light guides and two ormore emission light guides. FIG. 1A also illustrates sealed vessel 500including port 205 for receiving probe member 185.

FIG. 1B is a schematic illustration of an optical platform useful in thesystem of the invention for measuring pH. Referring to FIG. 1B, opticalplatform 180 includes excitation optics 280, first emission optics 380,and second emission optics 480. Excitation optics 280 include lightsource 282, collimating lens 284, filter 286, focusing lens 288, andexcitation light waveguide 290. First emission optics 380 includedetector 382, focusing lens 384, filter 386, collimating lens 388, andfirst emission light waveguide 390. Second emission optics 480 includesdetector 482, focusing lens 484, filter 486, collimating lens 488, andsecond emission light waveguide 490. Excitation light guide 290, firstemission light waveguide 390, and second emission light waveguide 490are housed in probe member 185.

The system's light source is effective in exciting the fluorescentspecies. Suitable light sources include light-emitting diodes, lasers,tungsten lamps, halogen lamps, xenon lamps, arc lamps, and hollowcathode lamps. In one embodiment, the light source is a light-emittingdiode emitting light in the range from 500 to 560 nm. A representativelight-emitting diode useful in the system of the invention is a greenultrabright Cotco 503 series LED commercially available from Marktech,Latham N.Y.

The collimating lens directs light (e.g., excitation light from thelight source or first and second emission light from the emission lightwaveguides) to the bandpass filter. Suitable collimating lenses includeBiconvex glass lenses and Plano-convex glass lenses. Representativecollimating lenses useful in the system of the invention are the TechSpec PCX lenses commercially available from Edmund Optics, Barrington,N.J. The excitation collimating lens is 12×36 (diameter by effectivefocal length in mm) and the first and second emission collimating lensesare 12×18.

The focusing lens focuses light from the bandpass filter to theexcitation light waveguide or from the bandpass filter to the detector.Suitable focusing lenses include Biconvex glass lenses and Plano-convexglass lenses. Representative focusing lenses useful in the system of theinvention are the Tech Spec PCX lenses commercially available fromEdmund Optics, Barrington, N.J. The excitation focusing lens is 12×18and the first and second emission focusing lenses are 12×15.

Filters are used in the optical platform to narrow the bandwidth oftransmitted light.

Suitable excitation filters include bandpass filters, shortpass filters,longpass filters, or a combination of short and long pass filters. Inone embodiment, the system uses a shortpass filter that passes light inthe range from about 370 nm to 540 nm. A representative excitationshortpass filter useful in the system of the invention is 540ASPcommercially available from Omega Optical, Brattleboro, Vt.

Suitable first emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 595 to 605 nm andhas a full width at half height of 10 nm. A representative firstemission bandpass filter useful in the system of the invention is600DF10 commercially available from Omega Optical, Brattleboro, Vt.

Suitable second emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 562 to 573 nm andhas a full width at half height of 10 nm. A representative secondemission bandpass filter useful in the system of the invention is568DF10 commercially available from Omega Optical, Brattleboro, Vt.

The excitation light waveguide transmits excitation light from the lightsource through the probe member to the fluorescent species. In oneembodiment, the excitation light waveguide includes one or more opticalfibers. In one embodiment, the excitation waveguide is a single opticalfiber. A representative fiber optic useful in the system of invention isRO2-534 commercially available from Edmund Optics, Barrington, N.J.

The first and second emission light waveguides transmit fluorescentemission from the fluorescent species through the probe member to thefirst and second emission detectors, respectively.

In one embodiment, the first emission light waveguide includes one ormore optical fibers. In one embodiment, the first emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thefirst emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system of invention is RO2-533commercially available from Edmund Optics, Barrington, N.J.

In one embodiment, the second emission light waveguide includes one ormore optical fibers. In one embodiment, the second emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thesecond emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system of invention is RO2-533commercially available from Edmund Optics, Barrington, N.J.

Suitable optical fibers useful in the system of the invention includeglass or plastic optical fibers from 0.2 to 2 mm diameter.

The system's first and second emission detectors are effective inmeasuring the first and second fluorescent emissions from thefluorescent species. Suitable detectors include photodiodes, PIN diodes,and photomultiplier tubes. In one embodiment, the first and secondemission detectors are photodiodes responsive in the range from 400 to800 nm. Representative photodiodes useful in the system of the inventioninclude BPW34 commercially available from Vishay Intertechnology,Malvern, Pa.

A representative probe member housing excitation and emission lightguides useful in the system of the invention is illustratedschematically in FIG. 2. As shown in FIG. 2, the light guides areoptical fibers. Referring to FIG. 2, probe member 185 houses excitationlight guide 290, a plurality of first emission light guides 390, and aplurality of second emission light guides 490. In the representativeprobe member shown in FIG. 2, there are four first emission light guides390, and four second emission light guides 490. The four first emissionlight guides can be considered to be a first channel (e.g., measuringthe first fluorescent emission from the fluorescent species) and thefour second emission light guides can be considered to be a secondchannel (e.g., measuring the second fluorescent emission from thefluorescent species). In the illustrated representative probe member,the fibers from each of the two sets of fibers alternate (i.e.,alternating fibers 390 and 490) around the central fiber (290). Thisconfiguration provides for evening out of “hot spots” so that lightcollected by the first set is similar to the light collected by thesecond set.

The relationship between the probe member housing theexcitation/emission light guides and the sealed vessel port isillustrated schematically in FIG. 3. Referring to FIG. 3, probe member185 is received by port 205. Port 205 includes window 210, which istransparent to excitation and emission wavelengths used in thefluorescent measurement. Excitation light emanating from light guide 290passes through window 210 and interrogates substrate 220 on which thefluorescent species is immobilized and which, in the operation of themethod of the invention, is in contact with the sample contained insealed vessel 200. Irradiation of substrate 220 results in excitation ofthe substrate-immobilized fluorescent species and fluorescent emissionfrom the fluorescent species. Emission from the fluorescent species isreceived by and transmitted through light guides 390 and 490 todetectors 382 and 482, respectively (see FIG. 1B). As noted above, thefluorescent species' first emission intensity and the second emissionintensity will depend on the pH of the sample.

A representative port assembly for introducing the substrate-immobilizedfluorescent species into a sealed vessel is illustrated in FIGS. 4A and4B. FIG. 4A illustrates the assembled port and FIG. 4B is an explodedview of the port assembly.

Referring to FIGS. 4A and 4B, port assembly 202 includes port 205 andtip 215. Port 205 is a cylinder terminating with window 210 and havingopening 212 for receiving probe member 185 (not shown). In oneembodiment, port 205 tapers from opening 212 to window 210 such that thedepth of insertion of probe member 185 into port 205 is predetermined bythe probe's diameter. In one embodiment, the depth of travel of 185 inassembly 202 is limited by a ledge (not shown). In one embodiment, theoptimal distance between probe and membrane was determined to be 2 mm orless. FIG. 21 illustrates the fluorescence intensity measured as afunction of distance between the probe and membrane. When inserted inthe port, the face of probe member 185 and window 210 are substantiallyparallel. Port 205 and tip 215 are adapted such that the port and tipare reversibly connectable. In one embodiment, port 205 includes annularinset 214 and tip 215 includes opening 216 defined by annular lip 218for receiving inset 214. In this embodiment, inset 214 has a diameterless than opening 216. It will be appreciated that the connectingrelationship between the port and tip can be reversed (i.e., port havingannular lip for receiving tip having inset). Lip 218 defines bed 222 forreceiving substrate 220, which is secured in port assembly 202 when port205 is connected to tip 215. Tip 215 includes aperture 224 in bed 222.Aperture 224 provides for contact of substrate 220 with a liquid samplecontained in a sealed vessel into which port assembly is introduced. Tip215 terminates with apex 226 that facilitates the introduction of portassembly 202 into a sealed vessel by puncture. FIG. 4C is a plan view oftip 215 illustrating bed 222 and aperture 224. In one embodiment, theassembly is made from Lexan HPS 1 1125 available from GE Polymerland,Pittsfield, Mass.

Representative sealed vessels incorporating the substrate-immobilizedfluorescent species are illustrated in FIGS. 5A and 5B. FIG. 5Aillustrates a sealed vessel into which a port assembly has been insertedby puncture. FIG. 5B illustrates a sealed vessel manufactured to includea port assembly.

Referring to FIG. 5A, sealed vessel 500 includes a plurality of vesselports 510. Port assembly 202 (including port 205, membrane 220, and tip215) resides in vessel port 510A after insertion. Vessel 500 remainssealed after insertion of port assembly 202. Vessel port 510A seals toport 205.

Referring to FIG. 5B, sealed vessel 500 includes a plurality of vesselports 510. Port assembly 232 (including port 205, membrane 220, and tip215) resides in vessel port 510A after vessel manufacture. A process formanufacturing a representative sealed vessel incorporating a portassembly is described in Example 1.

A representative port assembly useful for incorporation into a sealedvessel during manufacture is illustrated in FIG. 6. The port assemblyuseful for incorporation during vessel manufacture is substantially thesame as the port assembly useful for introduction into a sealed vesselillustrated in FIG. 4, except that the assembly useful in vesselmanufacture need not include, and preferably does not include, a featurefor puncturing the vessel. Referring to FIG. 6, port assembly 232includes port 205 and tip 235. Port 205 is a cylinder terminating withwindow 210 and having opening 212 for receiving probe member 185 (notshown). In one embodiment, port 205 tapers from opening 212 to window210 such that the depth of insertion of probe member 185 into port 205is predetermined by the probe's diameter. When inserted in the port, theface of probe member 185 and window 210 are substantially parallel. Port205 and tip 235 are adapted such that the port and tip are reversiblyconnectable. In one embodiment, port 205 includes annular inset 214 andtip 235 includes opening 216 defined by annular lip 218 for receivinginset 214. In this embodiment, inset 214 has a diameter less thanopening 216. It will be appreciated that the connecting relationshipbetween the port and tip can be reversed (i.e., port having annular lipfor receiving tip having inset). Lip 218 defines bed 222 for receivingsubstrate 220, which is secured in port assembly 202 when port 205 isconnected to tip 235. Tip 235 includes aperture 224 in bed 222. Aperture224 provides for contact of substrate 220 with a liquid sample containedin the sealed vessel.

Fluorescent Species Having pH-Dependent Emission.

The method and system of the invention for measuring pH uses afluorescent species having pH-dependent fluorescent emission. Thefluorescent species has a first emission intensity at a first wavelengthand a second emission intensity at a second wavelength, the first andsecond emission intensities being characteristic of pH in theenvironment of the fluorescent species. The ratio of the first andsecond emission intensities provides pH measurement. It is appreciatedthat fluorescent emission occurs as a wavelength band having a bandmaximum that is referred to herein as the emission wavelength.

In one embodiment, the separation between the first wavelength and thesecond wavelength is at least about 40 nm. In one embodiment, theseparation between the first wavelength and the second wavelength is atleast about 30 nm. In one embodiment, the separation between the firstwavelength and the second wavelength is at least about 20 nm. Using 10nm HBW filters, the separation is at least about 30 nm. Preferably, thesystem of the invention achieve fluorescence signal separation byremoving any emission band overlap by 10E5 or more.

The method and system of the invention for measuring pH are not limitedto any particular fluorescent species, nor any particular pH range. Themethod and system of the invention is operable with any fluorescentspecies having pH-dependent properties that can be excited and itsemission measured. The range of pH measurable by the method and systemof the invention can be selected and is determined by the pH-dependentproperties of the fluorescent species.

In addition to their pH-dependent properties noted above, suitablefluorescent species include those that can be substantially irreversiblyimmobilized on a substrate. The fluorescent species can be covalentlycoupled to the substrate or non-covalently associated with thesubstrate.

Suitable pH-dependent fluorescent species include those known in theart. Representative fluorescent species having suitable pH-dependentproperties include fluorescein derivatives including naphthofluoresceincompounds, seminaphthofluorescein compounds (e.g., SNAFL compounds), andseminaphthorhodafluor compounds (e.g., SNARF compounds). These compoundshave advantages associated with their long wavelength emission, which isless susceptible to potential interfering light absorbing substances inblood. These compounds also have relatively long wavelength absorbancemaking them particularly suitable for excitation by commerciallyavailable LED light sources. Another compound having suitable pHdependent behavior is HPTS, 8-hydroxy-1,3,6-pyrenetrisulfonic acid.Although the compound has desired ratiometric pH properties, excitationis optimal at short wavelength (403 nm) where strong LED light sourcesare not commercially available. Representative SNAFL and SNARF compoundsuseful in the method and system of the invention are described in U.S.Pat. No. 4,945,171. Molecular Probes (now Invitrogen, Eugene, Oreg.)sells CNF, SNAFL, SNARF fluors with conjugatable carboxylic acid linkergroups, see, for example, Molecular Probes Handbook (Ninth Edition) byR. P. Haugland, Chapter 21 “pH indicators” pages 829-847. EpochBiosciences (now Nanogen, Bothell, Wash.) sells EBIO-3 with a propanoicacid linker. Whitaker et al. (Anal. Biochem. (1991) 194, 330-344) showedthe synthesis of a number of SNAFL compounds. Wolfbeis et al. (MikrochimActa (1992) 108, 133-141) described the use of CNF and aminocelluloseconjugates. The earliest reference to the SNAFL family of compounds isWhitaker et al. (1988) Biophys. J. 53, 197a. A related dye in the CNFfamily is VITABLUE, a sulfonenaphthofluorescein derivative (Lee et al(1989) Cytometry 10, 151-164) having a pKa of 7.56. A CNF analog withbromine substituents at each carbon adjacent to a phenol (pKa 7.45) hasa pKa that is 0.54 pKa units lower than their measured pKa for CNF (pKa7.99). Lee et al. note that “true” pKa values are difficult to determinefor these compounds. A method for pKa determination is described inExample 3. SNAFL-1 (literature pKa ˜7.8) free acid had a pKa of 7.6 inthat fluorescence-based assay.

The structures of seminaphthofluorescein compounds (SNAFL-1 and EBIO-3)useful in the method and system of the invention are illustrated below.

The numbering scheme describes position of attachment of linkermolecules. These compounds have carboxylate linking groups suitable forconjugation to carrier proteins, as described below. For conjugation,the reactive N-hydroxysucinimide (NHS) ester of SNAFL-1 (commerciallyavailable from Molecule Probes, Inc., Eugene, Oreg.) can be used.Conjugation to lysine residues in human serum albumin (HSA) gave desiredSNAFL/HSA conjugates. Carbodiimide activation of EBIO-3 gave a reactiveintermediate that was efficiently conjugated to human serum albumin.

Representative naphthofluorescein and seminaphthofluorescein compoundsuseful in the method and system of the invention are illustrated in FIG.7.

The SNAFL compounds are commercially available from Molecular Probes,Inc., Eugene, Oreg. The SNAFL compounds can be readily synthesizedaccording to general procedures that have been published (see, forexample, U.S. Pat. No. 4,945,171).

The preparation of a representative 2-chloro substituted SNAFL compoundis shown below.

The compound can be prepared by condensation of 1,6-dihydroxynaphthalenewith the diacid substituted 4-acylresorcinol in the presence of adehydrating acid or Lewis acid catalyst, such as zinc chloride.

The preparation of SNAFL compounds having propionic acid linkers isdescribed in U.S. patent application Ser. No. 11/022,039, incorporatedherein by reference in its entirety. A representative SNAFL compoundshaving a propionic acid linker, EBIO-3, is commercially available fromNanogen, Bothell Wash.

The emission spectra as a function of pH of representative fluorescentspecies (i.e., SNAFL-1 and EBIO-1) useful in the method and system ofthe invention are illustrated in FIGS. 8 and 9, respectively. FIG. 8illustrates the emission spectra of SNAFL-1 in 50 mM potassium phosphatebuffer as a function of pH (pH 6.0 to 10.0) (excitation at 540 nm).Referring to FIG. 8, the response at pH 6-7 is relatively poor(pKa=7.6). FIG. 9 illustrates the emission spectra of EBIO-3 in 50 mMpotassium phosphate buffer as a function of pH (pH 6.0 to 10.0)(excitation at 545 nm). Referring to FIG. 9, the response at pH 6-7 isrelatively good (pKa=6.6). Spectral properties and pKa data for theSNAFL analogs illustrated in FIGS. 7A-7E are summarized in Table 1.

TABLE 1 pH-Sensitive absorbance and emission of SNAFL analogs.Absorbance Absorbance Emission λmax λmax Emission λmax Compound (acid)(base) λiso (base) pKa SNAFL-1 482, 510 nm 540 nm 585 nm 620 nm 7.6SNAFL-2 485, 514 547 590 630 7.6 EBIO-1 496, 519 545 560 620 6.5 EBIO-2506, 538 572 590 645 7.8 EBIO-3 480, 509 534 560 610 6.6

Referring to Table 1, absorbance and emission spectra were obtained at10 μM SNFL analog. Absorbance was measured at pH 6, 8, and 10: acid (pH6) gave two bands of similar absorbance; pH 10 gave a single λmax(base). The emission spectra were determined by excitation at theabsorbance λmax (base). The wavelength where emission spectra crossed isreported as λiso. The emission λmax was measured at pH 10. pKa wasdetermined from fluorescence emission spectra. EBIO-1 and EBIO-3 weremore sensitive to changes at pH ˜6.5. The other analogs were moresensitive at pH ˜8.

Benzo[c]xanthene Linker Compounds

In another embodiment, the fluorescent species is a benzo[c]xanthenelinker compound.

Representative benzo[c]xanthene linker compounds include compounds offormulae (I), (II), and (III), their salts, active esters, acid/baseforms, and tautomers.

In one embodiment, the benzo[c]xanthene linker compound has formula (I):

These benzo[c]xanthene linker compounds are exemplified by EBIO-3 orBCSI-3 (X₀ at 2 position is Cl, X₀ at 4 position is H). All other X andR=H. For EBIO-3 the linker arm is a C3-alkylcarboxylic acid (which maybe in the form of a lactone).

In another embodiment, the benzo[c]xanthene linker compound has formula(II):

The benzo[c]xanthene linker compounds of formula (II) differ from thoseof formula (I) by the position of the linker arm.

In a further embodiment, the benzo[c]xanthene linker compound hasformula (III):

The benzo[c]xanthene linker compounds of formula (III) differ from thoseof formulae (I) and (II) by the position and the nature of the linkerarm. The benzo[c]xanthene linker compounds of formula (III) areexemplified by BCSI-1 (X₀ at 2 position is Cl, X₀ at 4 position is H).All other X and R=H. In this embodiment, the linker arm is a carboxylicacid group at either position 5 or 6.

For the benzo[c]xanthene linker compounds of formulae (I), (II), and(III), substituents X₀-X₄, R_(1′), R_(1″), and R₁-R₄ are as describedbelow.

At least one of the X₀ groups is a halogen (e.g., chloro).

R_(1′), R_(1″), R₁, R₂, R₃, and R₄ are each independently selected fromhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl,(C₁-C₈)alkylthio and (C₁-C₈)alkoxy, aryl, and heteroaryl; X₁, X₂, X₃,and X₄ are each independently selected from the group consisting ofhydrogen, halogen, cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl,aryl(C₁-C₄)alkyl, heteroaryl, SO₃H, and CO₂H; wherein the alkyl portionsof any of R_(1′), R_(1″), R₁, R₂, R₃, and R₄, and X₁, X₂, X₃, and X₄ areoptionally substituted with halogen, carboxy, sulfo, amino, mono- ordialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkylportions of the substituents have from 1 to 6 carbon atoms; and whereinthe aryl or heteroaryl portions of any of R_(1′), R_(1″), R₁, R₂, R₃,and R₄, and X₁, X₂, X₃, and X₄ are optionally substituted with from oneto four substituents selected from the group consisting of halogen,cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C₁-C₆)alkylamino,(C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy.

Each of the benzo[c]xanthene linker compounds of formulae (I), (II), and(III) include a linker arm. As used herein, for the compounds offormulae (I) and (II), the term “linker arm” refers to a group of atomsthat includes a functional group capable of reaction with a nucleophilicsite on a protein, other macromolecule, or solid surface. Suitablefunctional (conjugatable) groups include those known in the art (e.g.,carboxylic acids and their reactive esters). In the examples, reactiveesters derived from carboxylic acid esters are described. However, itwill be appreciated that other electrophilic groups capable of reactionwith nucleophilic sites on proteins, other macromolecules, and solidsurfaces are within the scope of the invention.

In certain embodiments, the linker arm has the formula:

—CH₂CH₂-L₁-NH-L₂-FG

wherein L₁ is a linker moiety intermediate the adjacent methylene group(CH₂) and the amine group (NH),

wherein L₂ is a linker moiety intermediate the amine group (NH) and thefunctional group (FG), and

wherein FG is a functional group reactive toward and capable ofcovalently coupling the fluorescent dye compound to a suitably reactivematerial.

Linker moiety L₁ serves as a spacer between the methylene group and thenitrogen atom. Linker moiety L₁ has a length not exceeding the length ofa normal alkyl chain of 25 carbons. Suitable linker moieties L₁ includefrom one to about fifty (50) atoms selected from carbon, nitrogen,oxygen, hydrogen, and halogen. Representative L₁ groups include alkylenegroups (e.g., —(CH₂)_(n)—, where n is 1-12), phenylene groups (e.g., o-,m-, and p-C₆H₄—), and alkylene oxide groups (e.g., ethylene oxide,—(CH₂CH₂O)_(m)—, where m is 1-5). In one embodiment, L₁ is —(CH₂)—.

Linker moiety L₂ serves as a spacer between the amine group (NH) and thefunctional group (FG). Linker moiety L₂ has a length not exceeding thelength of a normal alkyl chain of 25 carbons. Suitable linker moietiesL₂ include from one to about fifty (50) atoms selected from carbon,nitrogen, oxygen, hydrogen, and halogen. Representative L₂ groupsinclude alkylene groups (e.g., —(CH₂)_(n)—, where n is 1-12), phenylenegroups (e.g., o-, m-, and p-C₆H₄—), and alkylene oxide groups (e.g.,ethylene oxide, —(CH₂CH₂O)_(m)—, where m is 1-5). Other suitable L₂groups include —C(=A₁)-L₁-, —C(=A₁)NH-L₁-, —C(=A₁)NH-L₁-NH—, wherein A₁is selected from O and S, and L₁ is as described above. In oneembodiment, L₂ is —C(═O)—(CH₂)_(n)—, where n is 2-6.

Representative FG groups include carboxylic acid groups, carboxylic acidactive esters (e.g., N-hydroxysuccinimide esters), maleimide groups,reactive carbamate and thiocarbamate groups, and α-haloacetamide groups(—NH—C(═O)—CH₂—X). Other suitable functional groups include groups thatare capable of coupling the cycloaddition (e.g., dienes and dienophilesto provide 4+2 cycloaddition products, and acetylenes and azides (clickchemistry)). In one embodiment, FG is a carboxylic acid group (—CO₂H) orits active esters (e.g., N-hydroxysuccinimide ester).

Carboxylic acid groups and carboxylic acid active esters are reactivetoward amino groups including the amino group of lysine residues inproteins and peptides, and primary amino groups introduced intooligonucleotide probes (—C(═O)—NH— linkage). Maleimide groups arereactive to sulfhydryl groups native to or introduced into protein,peptide, and oligonucleotides (—N[C(═O)CH₂CHC(═O)]—S— linkages).Reactive carbamate and thiocarbamate groups are reactive toward aminogroups to provide urea (—NH—C(═O)—NH—) and thiourea (—NH—C(═S)—NH—)linkages. α-Haloacetamide groups are reactive toward thiol groups toprovide —NH—C(═O)—CH₂—S— linkages. Functional groups capable ofconjugation through cycloaddition include dienes (e.g., furans) anddienophiles (e.g., alkenes and alkynes) that react to form 4+2cycloaddition linkages. The linker arm can be modified to include eithera diene or dienophile reactive toward a dienophile and diene,respectively, native to or incorporated into the complementary reactivematerial (e.g., biomolecule). Click chemistry can also be utilized forconjugation. The linker arm can be modified to include either a suitableacetylene (e.g., H—C≡C—R) or azide (e.g., R′—N═N⁺═N⁻) reactive toward anazide or acetylene, respectively, native to or incorporated into thecomplementary reactive material (e.g., biomolecule).

In one embodiment, the linker arm has the formula:

—(CH₂)_(n)—NHC(═O)—(CH₂)_(m)—CO₂H

wherein n is an integer from 1 to 12 and m is an integer from 1 to 12.In certain embodiments, n is an integer from 1 to 4. In certainembodiments, m is an integer from 1 to 4. In one embodiment, n is 3 andm is 2.

The preparation of a representative fluorescent species, BCSI-3, isdescribed in Example 12. FIG. 23 illustrates the synthesis of arepresentative 2-halo seminaphthofluorescein compound (BCSI-3) useful inthe method and system of the invention.

The absorbance spectra of two representative fluorescent species (EBIO-3and BCSI-3) are compared in FIGS. 24-26. FIG. 24 compares the absorbancespectra of two representative fluorescent species (EBIO-3 and BCSI-3) atpH 9.5 (borate buffer). FIG. 25 compares the absorbance spectra of tworepresentative fluorescent species (EBIO-3 and BCSI-3) at pH 7.4(phosphate buffered saline). FIG. 26 compares the absorbance spectra oftwo representative fluorescent species (EBIO-3 and BCSI-3) at pH 4.5(acetate buffer).

The pKa of the two representative fluorescent species (EBIO-3 andBCSI-3) are comparable. FIG. 27 illustrates the pKa determination of arepresentative fluorescent species (EBIO-3) by plotting peak absorbance(530 nm) as a function of pH. FIG. 28 illustrates the pKa determinationof a representative fluorescent species (BCSI-3) by plotting peakabsorbance (530 nm) as a function of pH.

The photostability of the two representative fluorescent species (EBIO-3and BCSI-3) are comparable. FIG. 29 compares the photodegradation of thetwo representative fluorescent species (EBIO-3 and BCSI-3) at pH 7.4(phosphate buffered saline) by plotting percent reduction in absorbanceat 530 nm as a function of exposure to light.

Fluorescent Species Conjugates for Substrate Immobilization.

For use in the method and system of the invention, the fluorescentspecies is immobilized on a substrate such that the fluorescent speciesis in contact with the sample, the pH of which is to be measured. Thefluorescent species can be immobilized on the substrate through the useof a material (e.g., macromolecular spacer material) having a strongassociative interaction with the substrate. The spacer material allowscovalent conjugation of the fluorescent species and provides largesurface area needed for efficient non-covalent immobilization to thesubstrate surface. In one embodiment, the spacer material is human serumalbumin (HSA) having ˜44 lysine residues available for covalentconjugation. HSA's densely charged molecular structure has a passivatingeffect when adsorbed to biomaterials. Other advantages include reducedfluorescence quenching, uniform environment for the conjugatedfluorophore, and availability in recombinant form (from yeast) so thereis no chance of infection (as with HSA from donors). HSA conjugates areeasily purified by ultrafiltration methods and form stable solutionsthat are easily characterized by absorbance and fluorescence assays todetermine the number of fluorophores per protein.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a protein or protein fragment. Suitableproteins include those that can be substantially irreversiblyimmobilized on the substrate. The protein can be covalently coupled tothe substrate or non-covalently associated with the substrate. Suitableproteins include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the protein.

Suitable proteins include human serum albumin (HSA), bovine serumalbumin (BSA), vonWillebrand's factor, kininogen, fibrinogen, andhemoglobin (no iron). Suitable proteins include proteins havingavailable lysine residues (for conjugation to the fluorophore) andmolecular weight sufficient to allow for immobilization efficiency tothe blot membrane. Other functional groups in the protein (likecysteine) could presumably be used for covalent bonding to suitablyreactive solid supports.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a polysaccharide. Suitable polysaccharidesinclude those that can be substantially irreversibly immobilized on thesubstrate. The polysaccharide can be covalently coupled to the substrateor non-covalently associated with the substrate. Suitablepolysaccharides include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the polysaccharide.

Suitable polysaccharides include dextrans, aminodextrans, heparin, andlectins.

In another embodiment, the fluorescent species is immobilized on thesubstrate through the use of dendrimeric structures. Suitabledendrimeric structures include those that can be substantiallyirreversibly immobilized on the substrate. The dendrimeric structurescan be covalently coupled to the substrate or non-covalently associatedwith the substrate. PAMAM dendrimers are commercially available as areother structural types and sizes.

In one embodiment, the fluorescent species is covalently coupled to aprotein to provide a fluorophore-protein conjugate that can beimmobilized on a substrate. In one embodiment, thefluorophore-polysaccharide conjugate is non-covalently associated withthe substrate.

In one embodiment, a fluorophore-protein conjugate is immobilized on asubstrate. In one embodiment, the fluorescent species is aseminaphthofluorescein and the protein is human serum albumin. In oneembodiment, the seminaphthofluorescein is SNAFL-1. The preparation ofSNAFL-1/HSA conjugates is described in Example 4. The fluorescentproperties of SNAFL-1/HSA conjugates are described in Example 5. In oneembodiment, the seminaphthofluorescein is EBIO-3. The preparation ofEBIO-3/HSA conjugates is described in Example 6. A schematicillustration of the coupling of EBIO-3 to HSA is illustrated in FIG. 10.The fluorescent properties of EBIO-3/HSA conjugates are described inExample 7.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (SNAFL-1/HSA, 1.6fluorophores per HSA) useful in the method and system of the inventionare illustrated in FIG. 11.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (EBIO-3/HSA, 1.92fluorophores per HSA) useful in the method and system of the inventionare illustrated in FIG. 12.

For the fluorophore-protein conjugate, the optimum fluorophore loadingwill vary depending on the particular fluorophore.

For SNAFL-1/HSA conjugates the fluorophore loading can vary from about0.01 to about 40 SNAFL-1/HSA. Low signal at 0.01 and fluorescentquenching at 40 fluorophores/HSA. In one embodiment, the SNAFL-1conjugate includes about 2 SNAFL-1/HSA.

For EBIO-3/HSA conjugates the fluorophore loading can vary from about0.01 to about 40 EBIO-3/HSA. In one embodiment, the EBIO-3 conjugateincludes about 2 EBIO-3/HSA.

In one embodiment, the fluorophore-protein conjugate is a conjugate of abenzo[c]xanthene linker compound of formulae (I), (II), or (III), and aprotein. The preparation of a representative fluorophore-proteinconjugate (BCSI-3/HSA) is described in Example 13. FIG. 30 is aschematic illustration comparing the preparation of two representativefluorophore-protein conjugates (EBIO-3/HSA and BCSI-3/HSA) useful in themethod and system of the invention.

FIG. 31 is a graph illustrating loading ratio of a representativefluorescent species (BCSI-3) to a representative protein (rHSA) as afunction of offering ratio (BCSI-3/rHSA). FIG. 32 compares fluorescentratio signal as a function of test buffer pH for representativefluorophore-protein conjugates (EBIO-3/HSA and BCSI-3/HSA conjugates)useful in the method and system of the invention.

Substrates for Fluorescent Species Immobilization.

In the method and system of the invention, the fluorescent species isimmobilized on a substrate. As noted above, the fluorescent species canbe directly immobilized on the substrate covalently or by non-covalentassociation or, alternatively, through the use of a material (e.g.,fluorophore-protein conjugate) that can be immobilized on the substratecovalently or by non-covalent association.

Suitable substrates substantially irreversible immobilized thefluorescent species. In the method of the invention, suitable substratesalso do not inhibit the contact of the liquid sample with thefluorescent species and do not impair or alter the pH measurement.

Representative substrates include membranes, such as microporousmembranes made of nitrocellulose, mixed esters of nitrocellulose andcellulose acetate, polyethylene terephthalate, polycarbonate,polyvinylidene fluoride and polyimide. Such materials are availablecommercially from Whatman S&S, Florham Park, N.J. and Millipore,Billerica Mass. Suitable membranes include membranes in which themicroporous structure is created by ion beam penetration such asmembranes commercially available from Oxyphen Gmbh, Dresden, Germanyunder the designation OXYPHEN. Charged nylon surfaces (Nytran) can alsobe used. Suitable membranes include plastic membranes in which themicroporous structure is made by injection molding the micropores intothe plastic such as the processes used by Åmic, Stockholm, Sweden.Emission intensity of SNAFL-1/HSA at pH 7 immobilized on various poresize mixed ester nitrocellulose cellulose acetate membranes is shown inFIG. 22.

Immobilization of representative fluorophore protein conjugates onmembranes is described in Examples 8 and 10. Example 8 describes theimmobilization of SNAFL-1/HSA conjugates. Example 9 describes thefluorescent properties of immobilized SNAFL-1/HSA conjugates. Example 10describes the immobilization of EBIO-3/HSA conjugates. Example 11describes the fluorescent properties of immobilized EBIO-3/HSAconjugates.

The emission spectra of a representative fluorophore-protein conjugate(SNAFL-1/HSA, 3.6:1) immobilized on Oxyphen and nitrocellulose as afunction of pH (pH response), as measured by the microwell assaydescribed in Example 9, are illustrated in FIGS. 13 and 14,respectively.

The emission spectra of a representative fluorophore-protein conjugate(EBIO-3/HSA, 2.0:1) immobilized on nitrocellulose, as described inExample 8, as a function of pH (6.0, 6.5, 7.0, 7.5, 8.0, and 10.0), asmeasured by the telescoping tube insert assay described in Example 11,are illustrated in FIG. 15. The large spread of emissions at 600 nm forthe pH 6 to 8 range indicates good fluorescence verses pH response.

The preparation of a representative membrane-immobilizedfluorophore-protein conjugate (BCSI-3/HSA/nitrocellulose) is describedin Example 14.

Ratiometric pH Method and System.

The method of the invention is a fluorescent wavelength-ratiometricmethod. In the method, the first and second fluorescent emissionintensities of the fluorescent species measured at first and secondemission wavelengths, respectively, are ratioed to provide pHinformation. The first emission wavelength varies with pH while thesecond emission wavelength is constant with pH and gives an internalcontrol for the fluorescent intensity. In one embodiment, a lookup tableis used to lookup a combination of the measured ratio, first and secondemission wavelength and determines its corresponding pH. In oneembodiment, a mathematical function of the ratio, first and secondemission wavelength is used to calculate the resulting pH.

FIG. 16 illustrates the data used in the method of the invention formeasuring pH. The emission spectra of a representativefluorophore-protein conjugate (EBIO-3/HSA, 2:1) immobilized onnitrocellulose at pH 7.0 is shown as measured by the telescoping tubinginsert assay. In this setup, the excitation bandpass filter was unableto completely remove the excitation light in the emission region asillustrated by the background signal measured on a blank nitrocellulosedisc. The full spectrum corrected for the background was multiplied bythe transmittance of the first and second hypothetical filters at eachwavelength and the area under the resultant curve was calculated to givea signal for the first and second wavelength. The center wavelengths andbandwidths of hypothetical filters were chosen such that the ratiometricproperties of the conjugate had the strongest relationship to the pH inthe region of interest.

FIG. 17 illustrates the results of the method of the invention forphosphate buffered saline (PBS), platelet poor plasma (PPP), andplatelet rich plasma (PRP) as measured by the telescoping tubing insertassay described in Example 11. The three curves represent the best fitrelationship between the measured pH and ratios for the three differentliquids.

FIG. 18 illustrates the correlation of pH results for three differentplasma samples obtained by the method and system of the invention asmeasured by the injection molded insert PVC tube assay described inExample 11. The relationship between the fluorescent signal and the pHhas an accuracy of about 0.1 pH units.

FIG. 19 illustrates stability of a representative substrate-immobilizedfluorophore conjugate of the invention (EBIO-3/HSA, 2:1) on mixed esternitrocellulose and cellulose acetate prepared by the soaking method andas measured by the leaching assay described in Example 10. The low levelof leaching is far below the toxic level for any compound.

Carbon Dioxide Measurement.

In another aspect, the present invention provides a device and methodfor measuring carbon dioxide concentration in a liquid sample. Thecarbon dioxide measuring method utilizes the pH measuring method andsystem described above. In the carbon dioxide measuring method anddevice, a substrate-immobilized fluorescent species as described aboveis in contact with a solution, the pH of which is responsive to carbondioxide level. In addition to being in contact with thesubstrate-immobilized fluorescent species, the solution having pHresponsive to carbon dioxide level is in contact with a liquid samplefor which the level of carbon dioxide is to be measured. The solutionhaving pH responsive to carbon dioxide level is isolated from the liquidsample for which the level of carbon dioxide is to be measured by aselectively permeable membrane. The membrane is permeable to gases(e.g., carbon dioxide) and impermeable to other materials (e.g.,liquids). Using the method of measuring pH described above, the pH ofthe solution responsive to carbon dioxide concentration in contact withthe substrate-immobilized fluorescent species is measured and correlatedwith the carbon dioxide level of the sample in contact with thatsolution.

The solution having pH response to carbon dioxide level is an aqueoussolution that includes an agent that is reactive toward carbon dioxideand changes the pH of the solution in response to carbon dioxideconcentration. Suitable agents that are reactive toward carbon dioxideand change the pH of the solution in which they are dissolved includebicarbonates, such as sodium bicarbonate.

The selectively permeable membrane isolates the solution having pHresponsive to carbon dioxide level from the liquid sample containingcarbon dioxide. The membrane is permeable to carbon dioxide andimpermeable to liquids and other solutes. In the method, carbon dioxidefrom the liquid sample passes from the liquid sample through themembrane and into the aqueous solution thereby reacting with the carbondioxide reactive agent to alter the pH of the aqueous solution. Suitableselectively permeable membranes include membranes made from silicone andPTFE.

FIG. 20 illustrates a representative device of the invention formeasuring carbon dioxide in a sealed vessel. Referring to FIG. 20,device 600 includes port assembly 610 including port 620 and tip 630.Port 620 is a cylinder terminating with window 622 and having opening624 for receiving probe member 185. When inserted in the port, the faceof probe member 185 and window 622 are substantially parallel. Port 620and tip 630 are adapted such that the port and tip are reversiblyconnectable. Substrate 640 including immobilized fluorescent species issecured within port 620 and tip 630. Tip 630 includes a chamber 645 forreceiving a solution having pH responsiveness to carbon dioxide. Chamber645 is defined by window 622, tip 630, and selectively permeablemembrane 650. Chamber 645 includes substrate 640, which is interrogatedby probe member 185.

A device for measuring carbon dioxide was assembled as described abovewith the membrane containing immobilized EBIO-3/rHSA conjugate (rHSA isrecombinant HSA). A layer of PARAFILM M, a blend of olefin-typematerials, was added under the membrane towards the tip. The membranewas hydrated with 5 ul of 35 mM carbonate buffer (pH 7.4), which wassealed within the assembly by the PARAFILM M and remained hydratedthroughout the assay. The assembly was subjected to 100% carbon dioxidegas by connection to the gas source with tubing and a “Y” adapter tobleed off the pressure. The assembly was subjected to the carbon dioxidefor an allotted period of time, allowed to acclimate to ambient airconditions, and repeated. The fluorescence was measured at each stage at568 nm and 600 nm after being excited at 525 nm. The results aresummarized below in Table 2 and reflect changes in fluorescence due tothe change in carbon dioxide concentration demonstrating that thefluorometric ratio method of the invention can also be used to calculatecarbon dioxide concentration. The PVC storage bags that are used forplatelet storage are somewhat gas permeable, and carbon dioxide isdirectly related to the measurement of pH.

TABLE 2 Carbon dioxide sensing results. Environmental EM at EM at RatioConditions 568 nm 600 nm (600/568) 15 min. at Ambient CO₂ 753 2184 2.9 5min. at 100% CO₂ 1179 2234 1.894 15 min. at Ambient CO₂ 833 2175 2.611 8min. at 100% CO₂ 1161 1930 1.662 60 min. at Ambient CO₂ 765 2184 2.854

The present invention provides a fluorescence-based pH indicator thatcan be easily inserted into the sampling ports of designed blood storagebags and interrogated using a fiber optic-based LED light source andphotodiode measurement system. This solid state system uses a“ratiometric” calibration method that accounts for variability influorescent signal strength due to interfering substances in blood thatmay interfere with the amount of excitation light that hits theindicator dye. The ratio of fluorescence intensities are measured at twowavelengths, one to detect the acid (protonated) isomer of the dye andone to detect the base (deprotonated) isomer.

To develop an accurate pH detector for platelet rich plasma, compoundshaving pKa of ˜6.6 are suitable, for example, 2-chloro substitution ofSNAFL compound lowers the pKa of the phenol from 7.6 to ˜6.6. Conjugatesof these compounds can be immobilized to various solid supports toprovide sensing pH membranes.

The present invention provides an inexpensive, easy to manufacture pHsensing membrane that gives accurate measurement of pH in plateletstorage bags at pH 6.5-7.5. In one embodiment, the invention uses aprotein conjugate (human serum albumin) of a 2-chloro substitutedratiometric fluorescent compound. The fluorophore:HSA ratio wasoptimized for performance when immobilized to a nitrocellulose blotmembrane. After drying on the membrane, the fluorophore:HSA conjugatehas very low leaching rates. Discs of this material are easily assembledinto holders for insertion into the sampling ports of platelet storagebags. The fluorescent membrane materials showed good pH response using agreen LED based fluorometer. In the method, two emission wavelengths forratiometric pH detection are measured with properly filtered photodiodeswith an accuracy of ˜0.1 units at the desired low pH threshold of 6.5.

Fluorescent probe molecules can be designed to be sensitive to a varietyof environments. The method and system of the invention describes theuse of pH-sensitive fluorophores. However, other environments can beinterrogated by the method and system of the invention modified toinclude environment-sensitive fluorophores other than pH-sensitivefluorophores. A variety of fluorescent probes that change fluorescentproperties as the molecular environment changes are commerciallyavailable. See, for example, Molecular Probes Handbook (9th Edition) byR. P. Haugland. Probes can be linked to albumins or other proteins andused to prepare substrates for interrogation as described in herein orusing other fluorescent-based methods. Examples of environment-sensitivefluorophores, systems, and methods include the following.

Nucleic acid detection: nucleic acid binding dyes change fluorescentproperties in the presence of DNA or RNA.

Enzyme substrates: proteins or peptides can be labeled with fluorescentdyes and fluorescent quenching molecules such that a fluorescent signalis generated in the presence of particular enzymes such as proteases(FRET detection).

Probes for lipids: lipophilic dyes can change fluorescent properties inthe presence of cell membranes or other lipid rich analytes.

Probes for oxygen: in addition to pH detection and carbon dioxidedetection, certain fluorescent molecules are sensitive to changes inoxygen concentration, for example, tris(2,2′-bipyridiyl)ruthenium(II)dichloride (RTDP).

Indicators for metal ions: fluorescent dyes that bind metals can changefluorescent properties upon binding calcium, magnesium, zinc, sodium,potassium, among other.

Glucose detection: certain lectins such as ConA bind glucose, andsuitably labeled lectins can be prepared as probes for glucose.

The following examples are provided for the purposes of illustrating,not limiting, the invention.

EXAMPLES Example 1 The Manufacture of a Vessel Incorporating aRepresentative Substrate-Immobilized Fluorescent Species

Referring to FIG. 5B, sealed vessel 500 is manufactured from PVC. PVCmaterial is compounded with a number of additives, for example,plasticizers, stabilizers, and lubricants. The formulation is used formaking bags and tubes. The compounded PVC is extruded through a die forconverting the plasticized material into sheet form. The extruded sheet,after slitting, is cut into the desired size and sent to the weldingsection. The donor and transfer tubings are made by extrusion of similarPVC compounds. The tubes are then cut to the appropriate length and sentto the welding section. The components, such as transfusion ports,needle covers, and clamp, are produced by injection molding. Thecomponents are ultrasonically cleaned and dried in a drying oven.

Welding.

The blood bags are fabricated by a high frequency welding technique.Sized PVC sheets are placed between electrodes and high frequency athigh voltage is applied. PVC gets heated very rapidly and sealing takesplace between electrodes. Transfusion ports and donor and transfertubing are kept in the appropriate position with the bag and welded toform an integral part of the blood bag system. For the manufacture of avessel incorporating a representative substrate-immobilized fluorescentspecies (FIG. 5B), an open tube is welded to provide port 510A. The tubecan be made of colored PVC to provide light protection for theimmobilized fluorescent species. Welded bags are trimmed. The portassembly 232 (FIG. 6) is manufactured from injection molded Lexan parts(205 and 235) and a 3.53 mm ( 9/64 inch) diameter nitrocellulose discwith immobilized fluorescent species (220). The port assembly is heldtogether by friction fit or can be glued in place. The port assembly isinserted in the open tube of port 510A. The port assembly is held in theport by friction fit or can be glued in place. The assembled bag andport assembly is sterilized and labeled for ultimate storage of plateletconcentrates.

Example 2 The Incorporation of a Representative Substrate-ImmobilizedFluorescent Species into a Sealed Vessel

Referring to FIG. 5A, sealed vessel 500 includes a plurality of vesselports 510. Port assembly 202 resides in vessel port 510A afterinsertion. The port assembly 202 (FIGS. 4A-4C) is manufactured frominjection molded Lexan parts (205 and 215) and a 3.53 mm ( 9/64 inch)diameter nitrocellulose disc with immobilized fluorescent species (220).The port assembly is held together by friction fit or can be glued inplace. The port assembly is inserted through the septum seal inside port510A by puncturing the seal with the spiked tip. Alternatively, the sealcan be pre-punctured with a separate spike tool. The insertion of theport assembly can be performed on either empty or platelet filled bags,but in either case, aseptic methods should be used to avoid possiblecontamination of the bag contents. The port assembly is held in the portby friction fit or can be glued in place. Vessel 500 remains sealed(leakproof) after insertion of port assembly 202 in port 510A.

Example 3 Fluorescence and pH Properties of Representative SNAFL AnalogspKa Determination

Instrumentation.

Fluorescence versus pH of various SNAFL free acids were compared usingan Ocean Optics USB2000 fiber optic spectrometer and a tungsten halogenlight source (part number HL-2000 FHSA). The light source was equippedwith a linear variable filter that allowed the wavelength and shape ofthe excitation beam to be adjusted. The excitation wavelength wasadjusted by using a blank cuvette to the absorbance max of thefluorophore (see Table 1). A cuvette holder (part number CUV-FL-DA) wasdirectly attached to the light source and a fiber optic cable directedemitted light to the spectrometer. Excitation conditions are reportedfor each fluorescence spectrum (3000 msec irradiation at the indicatedwavelength). Spectral data were collected on a personal computer usingthe Ocean Optics software and overlays of different spectra werecaptured.

Sample Preparation.

SNAFL-1 was purchased as the free carboxylic acid from Molecular Probesin a 1 mg vial. 0.3 mL of isopropyl alcohol and 0.7 mL of water wasadded to make a 1 mg/mL solution. A molecular weight (MW) of 426 forSNAFL-1 was used to calculate molarity (SNAFL-1=2.35 mM). 4.25 uL ofthis solution was diluted to 1 mL with various 50 mM phosphate buffersto give 10 micromolar solutions with pH 6-10. 10 micromolar solutions ofSNAFL-2 (MW=460) were prepared in a similar fashion. EBIO-1 (MW=523),EBIO-2 (MW=627), and EBIO-3 (MW=489) were obtained as bulk compoundsfrom Epoch Biosciences. 1.6 mg of each solid powder was carefullyweighed out and dissolved in 3.2 mL of 40% isopropyl alcohol to give 0.5mg/mL solutions. Emission spectra were obtained for the various SNAFLand EBIO compounds at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 8.0 and 10.0.Examples of overlayed fluorescence emission spectra are shown in FIG. 8(SNAFL-1) and FIG. 9 (EBIO-3). All spectra showed an isosbesticwavelength where all emission spectra overlap (See Table 1). This is acharacteristic of ideal ratiometric performance with no competingfluorescent structures other than those shown above (lactone, naphthol,naphtholate).

pKa Calculations.

The pH at which two molecular species (tautomers) are equallyrepresented is defined as the pKa. There are many variables that canaffect pKa and methods for measurement are difficult since thestructures have overlapping absorbance. Therefore direct comparisonsfrom the literature can vary slightly. The calculations contained hereinare based on the assumption that, at pH 10, only the trianionicnaphtholate structure is present. The intensity of fluorescence at theemission maxima is divided by 2, and pH of the intersecting pH curve iscalculated by interpolation between the nearest 2 curves. The pKa of the2-chloro substituted EBIO compounds is significantly lower than theother analogs as shown in Table 1.

Example 4 The Preparation of Representative Fluorophore-ProteinConjugates SNAFL-1/HSA

Human serum albumin (HSA) was purchased from Sigma (catalog #A-8763) as100 mg of lyophilized powder. SNAFL-1 NHS ester was purchased fromMolecular Probes as a mixture of the 5 and 6 isomers. A solution of 10mg (0.15 micromoles) of HSA in 1 mL of pH 8.56 sodium bicarbonate (0.1M) was prepared. A solution of 1 mL (1.91 micromoles) of the NHS esterin 0.1 mL of dimethylsulfoxide was prepared. 0.3 mL aliquots of the HSAsolution were transferred to a 1.6 mL Eppendorf tubes and variousoffering ratios of the NHS ester solution were added: tube 1, 11.8microliters (5 equivalents); tube 2, 23.6 microliters (10 equivalents),tube 3, 47.1 microliters (20 equivalents). The deep red solutions werevortexed and allowed to stand in the dark for at least one hour. The 5:1conjugate from tube 1 was purified by gel filtration chromatography on a0.5×20 cm column packed with Sephadex G-15 and pH 7.4 phosphate bufferedsaline (PBS). The conjugate was isolated as a fast moving red/orangeband in PBS and diluted to 0.75 mL with PBS to give a 4 mg/mL solutionof the protein conjugate. Most of the color eluted with the conjugate,but some small molecular weight (orange) impurities remained on top ofthe column. The column was clean enough to be re-used for purificationof the 10:1 and 20:1 conjugates. Each was eluted in PBS and diluted to0.75 mL to give ˜4 mg/mL solutions (60 micromolar based on HSAcomponent). The red solutions were stored refrigerated and protectedfrom light. 1 micromolar solutions of each SNAFL-1/HSA conjugate wereprepared and analyzed by UV-vis spectra using a Beckman DU640Bspectrometer. Each spectrum showed absorbance maxima at 490 and 521 nmat pH 7 as expected for the acid form of SNAFL-1 conjugates. Therelative absorbance showed the expected change in absorbance withdifferent SNAFL:HSA offering ratio. A 10 micromolar solution of SNAFL-1acid (obtained from Molecular Probes) at pH 7 was used as a standard tomore accurately determine the average loading of SNAFL-1 per each HSAconjugate preparation. Using this assay, the 5:1 conjugate had 4.1fluors/HSA, the 10:1 conjugate had 6.4 fluors/HSA, and the 20:1conjugate had 11.2 fluors/HSA.

Example 5 The Fluorescent Properties of RepresentativeFluorophore-Protein Conjugates SNAFL-1/HSA

Relative fluorescence of various SNAFL-1/HSA conjugates and SNAFL-1 freeacid were compared using an Ocean Optics USB2000 fiber opticspectrometer and a tungsten halogen light source (part number HL-2000FHSA). The light source was equipped with a linear variable filter thatallowed the wavelength and shape of the excitation beam to be adjusted.A cuvette holder (part number CUV-FL-DA) was directly attached to thelight source and a fiber optic cable directed emitted light to thespectrometer. Excitation conditions are reported for each fluorescencespectrum (3000 msec irradiation at the indicated wavelength). Spectraldata were collected on a personal computer using the Ocean Opticssoftware and overlays of different spectra were captured. A comparisonof various loading levels of SNAFL-1/HSA showed that 4.1 to 1.6 SNAFL-1molecules gave about the same fluorescent signal. Higher loading orlower loading conjugates gave lower signals.

Emission spectra were obtained for 10 micromolar solutions in potassiumphosphate buffer. Excitation was at 540 nm. Emission maximum at 620 nmwas observed for the base form of SNAFL-1 (pH 10). As expected,intensity of 620 nm fluorescence decreased as pH decreased. Anisosbestic point at 585 nm, where fluorescence remained constant at allpH, was observed. Response was good at about pH 8, but poor between pH6-7.

Spectra obtained for a 2.5 micromolar solution of a representativeSNAFL-1/HSA conjugate (1.6 SNAFL-1/HSA) showed improved pH response forpH 6-7 (see FIG. 11). The Ocean Optics halogen light source was equippedwith a 532 nm interference filter (Edmund Optics, Barrington, N.J.) andthis allowed the fluorescent isosbestic point at 572 nm for pH 6-7 to bedetected. Emission maximum at 620 nm was observed for the base form ofSNAFL-1 (see pH 10 curve). As expected, intensity of 620 nm fluorescencedecreased as pH decreased. In comparison to the free SNAFL-1 carboxylicacid (see FIG. 8) improved response for pH 6-7 was observed for the HSAconjugate. A red shift of the pH 8 and 10 curves from the isosbesticwavelength was observed, indicative of other competing molecularstructures involving the fluorescent species. This non-ideal behaviormay be eliminated by use of a longer linker structure or a morehydrophilic linker structure between the fluorescent dye and the HSAspacer.

Example 6 The Preparation of Representative Fluorophore-ProteinConjugates EBIO-3/HSA

Method A.

A 0.1 M stock solution of EDC (Sigma/Aldrich Chemical Co., St. LouisMo.) was prepared by dissolving 6.2 mg of EDC in 0.2 mL of DMF and 0.123mL of 50 mM phosphate buffer (pH 5.8). 1.0 mg of EBIO-3 acid (Nanogen,Bothell, Wash.) was dissolved in 0.102 mL of DMF to give a 20 mMsolution. 3.0 mg (0.045 micromoles) of HSA (Sigma/Aldrich Chemical Co.,St. Louis, Mo.) was dissolved in 0.3 mL of pH 8.5 sodium bicarbonate ineach of two 1.7 mL Eppendorf tubes. 0.1M EDC (0.045 mL) was added to 20mM EBIO-3 (0.045 mL, 0.9 micromoles) in a separate Eppendorf tube andthis was added to one of the HSA tubes to give an EBIO-3:HSA offeringratio of 20:1. An offering ratio of 5:1 was used in the other HSA tubeby adding a premixed solution of 0.0225 mL of EDC (0.1 mM) and 0.0225 mLof EBIO-3 (20 mM). The homogeneous dark red HSA conjugate solutions wereincubated at room temperature in the dark. After 21 hours, each of theHSA conjugates was purified on a G15 Sephadex column as described abovefor the SNAFL conjugates (Example 4). Some unreacted EBIO-3 acidremained at the top of the column (especially for the 20:1 offeringratio), but was cleanly separated from the desired protein conjugatethat eluted first as a pink fraction in ˜0.5 mL of pH 7.4 buffer. Eachof the purified conjugates was diluted to 0.75 mL with pH 7.4 PBS togive 4 mg/mL solutions (0.06 mM). The red solutions were storedrefrigerated and protected from light. 1 micromolar solutions of eachEBIO-3/HSA conjugate were prepared at pH 7.4 and analyzed by UV-visspectra using a Beckman DU640B spectrometer. The free EBIO-3 acid (10micromolar) spectrum had absorbance maximum at 534 nm, the 20:1conjugate had absorbance at 538 nm and the 5:1 conjugate had maximum at545 nm. The spectra showed the expected increase in absorbance withincreasing EBIO-3:HSA offering ratio. Using this EBIO-3 acid as astandard, the 20:1 conjugate had 5.07 EBIO-3:HSA and the 5:1 offeringhad 1.92 EBIO-3:HSA. The coupling efficiency was somewhat lower than forthe SNAFL/HSA conjugates of Example 4 (the 20:1 conjugate had 11.2fluors/HSA and the 5:1 offering had 4.1 fluors/HAS). The EDC couplingmethod was suitably efficient and reproducible.

Method B.

A 0.1 M solution of EDC (Sigma/Aldrich Chemical Co., St. Louis, Mo.) isprepared by dissolving 6.0 mg of EDC in 0.194 mL of DMF and 0.118 mL of50 mM PBS (pH 7.4). 3.0 mg of EBIO-3 acid (Nanogen, Bothell, Wash.) isdissolved in 0.306 mL of DMF to give a 20 mM solution. The two solutionsare combined in the EBIO-3 solution container and incubated at roomtemperature for one hour in the dark. 75.0 mg (1 micromole) of liquidrecombinant HSA (rHSA) from yeast (Delta Biotechnology, Ltd.,Nottingham, UK) is mixed with 7.5 mL of pH 8.5 sodium bicarbonate in a15 mL conical tube. The entire contents of the EBIO-3/EDC solution arecombined with the rHSA solution and incubated at room temperature in thedark for 15-20 hours. The rHSA/EBIO-3 conjugate is purified using theAmicon stirred ultrafiltration cell system and a YM10 membrane(Millipore, Bedford, Mass.). A 50 mM PBS (pH 7.4) is used as the washsolution. After purification, the protein concentration of the conjugateis measured using the BCA™ Protein Assay (Pierce, Rockford, Ill.). Analiquot of the conjugate is diluted to 1 mg/ml with 50 mM PBS (pH 7.4)based on its BCA determined protein concentration. The 1 mg/ml aliquotof conjugate, the last milliliter of PBS effluent, an aliquot of the 50mM PBS (pH 7.4), and an aliquot of the EB3 Standard (15 mM EBIO-3solution in DMF and 50 mM PBS (pH 7.4)) are analyzed via an absorbancescan utilizing Bio-Tek's Synergy HT plate reader. The scan is taken on300 microliters of each of the above mentioned samples in a black,96-well, clear, flat bottom plate, scanned from 450 nm to 650 nm. Theirmax peaks are recorded and used to determine purity and quality of theconjugate.

Example 7 The Fluorescent Properties of RepresentativeFluorophore-Protein Conjugates EBIO-3/HSA

Fluorescence spectra were obtained for 2.5 micromolar solutions of thetwo EBIO-3/HSA conjugates prepared as described in Example 6 (Method A).The conjugates showed improved pH response for pH 6-7 (see FIG. 12 foroverlayed spectra for the 1.92:1 EBIO-3/HSA conjugate). The Ocean Opticshalogen light source was equipped with a 532 nm bandpass filter (EdmundOptics, Barrington N.J.) and this allowed the fluorescent isosbesticpoint at ˜565 nm for pH 6-7 to be detected. Emission maximum at 605 nmwas observed for the base form of SNAFL-1 (red trace, pH 10). Asexpected, intensity of 605 nm fluorescence decreased as pH decreased. Incomparison to the SNAFL-1/HSA conjugate (see FIG. 11) improved responsefor pH 6-7 was observed for the EBIO-3/HSA conjugate. A red shift of thepH 8 and 10 curves from the isosbestic wavelength was observed,indicative of other competing molecular structures involving thefluorescent species, but was of smaller magnitude than for theSNAFL-1/HSA conjugate.

Example 8 Immobilization of Representative Fluorophore-ProteinConjugates SNAFL-1/HSA

Fluorophore-protein conjugates and fluorophore-carbohydrate conjugateswere immobilized on either nitrocellulose or capillary pore membranesusing the following general method. Fluorescein labeled dextrans with“fixable” lysine residues were obtained from Molecular Probes. Thesedextrans had a molecular weight of about 10,000 1.8 fluorophores perconjugate, and 2.2 lysines per conjugate and are sold under the tradename “Fluoro-Emerald”. Fluorescein labeled bovine serum albumin (BSA)was also obtained from Molecular Probes and had 4.5 fluors perconjugate. Various SNAFL-1/HSA conjugates were prepared as described inExample 4. Nitrocellulose membranes were obtained from Schleicher andSchuell under the trade name PROTRAN. Pore diameter was reported as 0.2microns. Capillary pore membranes made from polyester films wereobtained from Oxyphen in a variety of pore sizes. 0.1 micron and 1.0micron pore size membranes were successfully used to immobilizefluorescein dextrans. Fluorescein/dextran, fluorescein/BSA andSNAFL-1/HSA conjugates were all successfully immobilized and thefluorescent properties of the SNAFL-1/HSA conjugates were fullycharacterized as described as follows.

General Immobilization Method.

SNAFL-1/HSA (2.5 SNAFL-1/HSA) on 0.1 micron pore diameter OxyphenMembrane Discs. Fluorescent HSA conjugates with a 2.5:1 SNAFL-1:HSAoffering ratio were prepared as described in Example 4 and diluted toprovide concentrations of 0.05, 0.2, 1.0 and 4 mg/mL in phosphatebuffered saline (PBS) (pH 7.4). 5 microliter drops were applied via a 20microliter pipettor to the center of pre-punched porous discs (¼ inchdiameter) that were laid on a bench top. The spotted discs were allowedto air dry (about 30 minutes) and then placed in separate desiccatorsovernight. The dried discs were washed in separate Eppendorf tubes with2×1 mL of PBS and allowed to soak overnight in 1 mL of PBS. The washeddiscs were stable in PBS solution (no degradation after 30 days).Alternatively the discs could be re-dried in desiccators and stored dry.The wet or dry stored discs had comparable fluorescent properties. Thediscs had fluorescent signals that were proportional to theconcentration of labeled macromolecule applied to each one as measuredby the fluorescence assay described in Example 9.

Example 9 The Fluorescent Properties of Representative ImmobilizedFluorophore-Protein Conjugates SNAFL-1/HSA

Microwell Assay of Fluorescent Macromolecular Conjugates on PorousMembrane Discs Using a Fiber Optic Spectrometer.

Fluorescent discs prepared as described in Example 8 were examined forfluorescent properties using the Ocean Optics fiber optic spectrometerdescribed in Example 5. The cuvette on the light source was replaced bya fiber optic reflectance probe which had 6 excitation fibers wrappedaround a single fiber that picks up the emitted light from the sampleand sends it to the spectrometer. The reflectance probe was threadedthrough a hole in a 12×12×18 inch black box with a lid on the front. Theprobe was clamped inside under a 1 cm square opening that allowed thetip of the probe to be positioned under a 96-well micro well plate(clear bottom black plate). The probe was tilted at a 30 degree angle toreduce reflected light entering the probe tip. The fluorescent disc ofinterest was placed in the bottom of a well and covered with 300microliters of the analyte solution of interest. The excitation lightsource was turned on long enough to position the disc of interest overthe tip of the reflectance probe, then the shutter was closed and theplate was covered with another box to shield the disc from ambientlight. Unless otherwise mentioned, the Ocean Optics software was set tocollect data with a 3000 msec integration time and 3 averages. A darkspectrum was captured with the shutter closed and used for allbackground subtracted readings during the assay. The shutter was thenopened and fluorescent reading of the disc was started. The graphicaldisplay on the computer screen gave real-time spectra after each 3000msec integration time. After the required 3 spectra were obtained (about10 seconds) the graphical display showed only subtle changes. At thispoint a snapshot of the displayed spectrum was captured and saved todisc for future processing. The shutter was closed, and the nextmicrowell experiment was set up. The same disc could be measuredmultiple times by exchanging the analyte solution in the microwells.Alternatively, different discs in different wells could be measured byre-positioning the microwell plate over the reflectance probe.

Fluorescent Loading of SNAFL-1/HSA Immobilized on Oxyphen Discs.

The microwell assay described above was used to compare therelative-fluorescence of SNAFL-1/HSA on Oxyphen discs. The excitationfilters in the halogen light source were set to a wavelength of 532 nmand a “wide open” bandpass position to maximize sensitivity of theassay. Reflectance of the excitation beam back into the detector fiberwas significant, and the wavelength position of the filter was adjustedto provide a “minimum” at 620 nm where the fluorescence from the baseform of SNAFL-1/HSA is greatest. The various concentrations ofSNAFL-1/HSA described in Example 8 were examined in separate microwellsin pH 7 potassium phosphate buffer (50 mM) as described above. Thespectra showed the ability to distinguish relative fluorescenceintensity of 4, 1, 0.2 and 0.05 mg/mL membranes at pH 7. All had signalgreater than background.

Relative Fluorescence Intensity of Various Amounts of SNAFL-1/HSA(2.5:1) Immobilized on Porous Oxyphen Discs at pH 7.

The fluorescent intensity was measured at 620 nm, the fluorescentmaximum of the base form of the fluorophore. Excitation used a wide opensetting on the halogen lamp that efficiently excites both acid and baseforms of SNAFL-1. The reflected light from the source (unmodified disc)had the lowest intensity spectrum. The spectrum of the 0.05 mg/mL discgave a small increase in fluorescence intensity. The 0.2 mg/mL disc, 1mg/mL disc, and 4 mg/mL disc showed stepwise increases in fluorescenceintensity. The fluorescence spectra of two 30 day PBS soaked sample (1mg/mL) from a different batch of membranes were essentially the same andshowed that membrane loading was reproducible from batch to batch, andthat the SNAFL-1/HSA conjugates did not dissociate significantly fromthe disc surface in PBS solution.

pH Dependent Fluorescence of SNAFL-1/HSA Immobilized on Oxyphen Discs.

The 1 mg/mL SNAFL-1/HSA discs described above were examined for pHdependent response in the microwell assay. A single disc was examined inpotassium phosphate buffers of pH 4, 5, 6, 7, 8, 9, and 10. The datashowed that these membrane discs had a wide dynamic range of pHmeasurement, but had more sensitive response at pH >6. The time betweenbuffer exchanges was 5 min, and there was no significant change inspectra after additional equilibration time. This showed that theresponse time for even dramatic changes in the pH environment of theimmobilized SNAFL-1/HSA conjugates is rapid.

“Crossover Assay” for Fluorescence Measurement of pH Using SNAFL-1/HSAOxyphen Discs.

The microwell assay described above was used to examine the fluorescentisosbestic properties of the discs. For this assay, the shutter assemblyin Ocean Optics halogen light source (part number HL-2000 FHSA) wasremoved, and two 532 nm bandpass filters (Edmund Scientific) wereinserted in the cavity using a special adaptor. This dramaticallyreduced the reflected background in the spectral region of interest(>550 nm). The data shown are for 4 mg/mL loading discs prepared withSNAFL:HSA (5:1) conjugate. The immobilized protein conjugate showedunusual pH vs. fluorescence properties in comparison to the solutionphase data. Instead of a fluorescent isosbestic point at 575 nm, therewas a stepwise increase in the fluorescent intensity as pH increased.The pH 10 spectrum showed the expected maximum at 620 nm, and crossedthe overlaid spectral curves obtained in pH 4, 6, 7 and 8 buffers. These“crossover points” were used as the basis for a sensitive assay todetermine pH of the membrane environment. Three different membrane discswere examined using this assay format on three different days. Thecrossover points were reproducible within 2 nm.

The 4 mg/mL discs (3.6:1 SNAFL-1:HSA) showed stepwise increase in pH 10“crossover”. The crossover was at 579 nm for pH 4. Three discs/threedifferent days gave the same result±2 nm. The crossover points were at592 nm (pH 6), 600 nm (pH 7a,b), and 611 nm (pH 8). The fluorescentmaximum at pH 10 was at 620 nm, similar to the solution phaseproperties.

Example 10 Immobilization of Representative Fluorophore-ProteinConjugates EBIO-3/HSA

Spotting Immobilization Method.

EBIO-3/HSA conjugate was prepared as described in Example 6 at a ratioof 2:1. Nitrocellulose membranes were obtained from Schleicher andSchuell under the trade name PROTRAN. The discs were treated in the sameway as the general immobilization method described in Example 5 using a4 mg/ml solution of EBIO-3/HSA.

Soaking Immobilization Method.

EBIO-3/HSA conjugate was prepared as described in Example 6 at a ratioof 2:1. Mixed ester nitrocellulose and cellulose acetate membranes wereobtained from Millipore under the product series TF. The EBIO-3/HSAconjugate is diluted to 0.2 mg/ml and 45 ml is added to a 9 cm disc ofthe membrane. The disc is agitated overnight at room temperature andprotected from light. The unbound conjugate is removed and the disc iswashed with two 1 hour washes and one overnight wash all with agitation.The disc is then desiccated and stored dry. Smaller discs are punchedfrom the 9 cm disc for studies.

Example 11 The Fluorescent Properties of Representative ImmobilizedFluorophore-Protein Conjugates EBIO-3/HSA

Telescoping Tubing Insert Assay of Fluorescent Macromolecular Conjugateson Porous Membrane Discs Using a Fiber Optic Spectrometer.

Fluorescent discs prepared as described in Example 10 were examined torelate the fluorescent properties to the liquid phase pH using the OceanOptics fiber optic spectrometer described in Example 9 with the dual 532nm filtered (Edmund Scientific) halogen light source (part numberHL-2000 FHSA). A holder for a 5/32 inch membrane disc was crafted with 4mm OD and 5 mm OD polystyrene telescoping tubing and an angled 0.015 inthick polystyrene window. The angled window was placed so that it heldthe membrane disc at a 60 degree angle relative to the tubing axis. Thisallows the fiber optic probe to be placed in one end of the tubing andinterrogate the disc on the other side of the window which is contactwith a liquid of a certain pH. Buffers of known pH values were placed incontact with the telescoping tubing inserts and discs made by thespotting immobilization method in Example 10 and fluorescent emissionsrecorded with the Ocean Optics software set to collect data with a 1000msec integration time and 3 averages.

For liquids with unknown pH values, a stirred and light protected vesselcontaining 5 telescoping tubing inserts and discs made by the soakingimmobilization method in Example 10, 50 mL of buffer or plasma, and acalibrated pH electrode (ROSS electrode/Orion 720a meter) was used tostudy the pH and fluorescent response of the fluorescent discs. Drops of1 N HCl or 1 M NaOH were added to create a range of pHs from liquidsstudied. Fluorescent spectra were collected through Ocean Optics macrosin Excel set to read for 1000 msec integration time and three averages.The spectra were analyzed using the modeled bandpass filters andratiometric method in Excel to obtain calibration curves for PBS,platelet poor plasma and platelet rich plasma.

Injection Molded Insert PVC Tube Assay of Fluorescent MacromolecularConjugates on Porous Membrane Discs Using a Custom OptimizedFluorescence Based pH Detector.

Injection molded polycarbonate parts were fashioned to fix thefluorescent discs to the fluorescence pH detector probe as pictured inFIG. 4. Membranes were prepared as described in the soakingimmobilization method in Example 10 and assembled into the plasticinsert. A 1 in long and 3/16 in ID PVC tube was placed on the spike endof the insert such that 250 ul of liquid was placed in the tube andcovered with parafilm to slow carbon dioxide desorption. A fluorescentmeasurement of the first and second wavelengths was taken and then thepH was read by a blood gas analyzer (Bayer 348). The pH of plasmasamples were adjusted by acid and base additions as in the telescopingtubing insert assay to create the range of pH data.

Example 12 The Preparation and Properties of a RepresentativeFluorescent Species BCSI-3

In this example, the preparation and properties of a representativefluorescent species, BCSI-3, is described. The preparation isillustrated schematically in FIG. 23.

General Procedures.

All TLC was run with Sigma-Aldrich silica gel plates (catalog #Z193275).¹H NMR were obtained on a Bruker 300 MHz spectrometer in dimethylsulfoxide—d₆) at room temperature. Chemical shifts (ppm) were referencedto dimethyl sulfoxide (2.49 ppm). All solvents, reagents and silica gelfor column chromatography were purchased from Sigma-Aldrich.

N-[3-2,4-Dimethoxyphenyl)prop-2-yn-1-yl]-2,2,2-trifluoroacetamide (2)

A solution consisting of anhydrous DMF (2.0 ml), anhydrous triethylamine(3.9 ml) and propargyl trifluoroacetimide (5.3 g, 35.1 mmol) wasdeoxygenated by bubbling a stream of argon through the solution for 20min. This solution was then added to a mixture of1-Iodo-2,4-dimethoxybenzene (4.9 g, 18.6 mmol), CuI (37 mg, 0.21 mmol)and tetrakis[triphenyl-phosphine]-palladium[0] (120 mg, 0.10 mmol). Theresulting mixture was stirred under an argon atmosphere for 24 hrs. Thereaction mixture was diluted with 50 ml of ethyl acetate and washed withwater (3×50 ml). The organic phase was dried over sodium sulfate,filtered and evaporated. The residue was purified by silica gelchromatography eluting with a gradient of 10-25% ethyl acetate inhexane. The product fractions were evaporated and the residue wascrystallized from methanol-water: 2.3 g (43% yield); TLC (50/50, ethylacetate/hexane), R_(f)=0.59; ¹H NMR (DMSO-d₆) δ 10.05 (1H, br s, N—H),7.29 (1H, d, J=8.4 Hz, aromatic-H), 6.59 (1H, d, J=2.4 Hz, aromatic-H),6.51 (1H, dd, J=8.4 & 2.4 Hz, aromatic-H), 4.25 (2H, s, methylene-H),3.79 and 3.78 (6H, 2×s, methoxy-Hs).

N-[3-2,4-dimethoxyphenyl)propyl]-2,2,2-trifluoroacetamide (3)

Absolute ethanol (40 ml) was carefully added to a 150-ml round-bottomflask containing 2 (2.2 g, 7.7 mmol) and 5% Pd/C (0.40 g) under an argonatmosphere. Ammonium formate (6.5 g, 103 mmol) was added and theresulting mixture was refluxed for 40 min. The reaction mixture wasfiltered through Celite and the filter cake was rinsed with 100 ml ofmethanol. The filtrate was evaporated to dryness and the residue wassuspended in 30 ml of water and extracted with ethyl acetate (2×40 ml).The pooled extracts were dried over sodium sulfate, filtered andevaporated affording a homogenous oil, which transformed into acrystalline solid upon overnight storage in a standard commercialfreezer: 2.0 g (89% yield). TLC (50/50, ethyl acetate/hexane),R_(f)=0.75.

N-[3-(2,4-Dihydroxyphenyl)propyl]-2,2,2-trifluoroacetamide (4)

A suspension of 3 (1.06 g, 3.6 mmol) was stirred in 15 ml of a 1Msolution of boron tribromide in methylene chloride for 45 min. at roomtemperature. Ice-cold methanol (5 ml) was carefully added drop-wise andthe resulting solution was evaporated to dryness. The residue waspurified by silica gel chromatography eluting with 50/50, ethylacetate/hexane. The pure product fractions were evaporated affording ahomogenous oil: 0.76 g (79% yield). ¹H NMR (DMSO-d₆) δ 9.39 (1H, br t,trifluoroacetimido, N—H), 9.11 and 8.96 (2H, 2×s, phenol-Hs), 6.80 (1H,d, J=8.5 Hz, aromatic-H), 6.27 (1H, d, J=2.5 Hz, aromatic-H), 6.13 (1H,dd, J=8.4 & 2.5 Hz, aromatic-H), 3.16 (2H, m, methylene-H), 2.40 (2H, t,J=7.5 Hz, methylene-H), 1.66 (2H, m, methylene-H).

2-(2,4-Dihydroxy-5-{3-[(trifluoroacetyl)amino]propyl}benzoyl)benzoicacid (5)

A mixture of 4 (0.56 g, 2.1 mmol), phthalic anhydride (0.32 g, 2.2 mmol)and aluminum chloride (0.80 g, 6.0 mmol) was stirred in anhydrousdichloroethane (under argon) for 20 hr. The dichloroethane solvent wasdecanted away and the solid residue was dissolved in 50 ml of ethylacetate and washed with 50 ml of water. The pH of the aqueous phase wasadjusted to approximately pH 3 by addition of acetic acid and thenextracted with ethyl acetate (2×50 ml). The combined organic extractswere dried over sodium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography eluting with a gradient of 25-0%hexane in ethyl acetate. The pure product fractions were evaporatedaffording a homogenous oil: 0.32 g (34% yield).

2-[5-(3-Aminopropyl)-2,4-dihydroxybenzoyl]benzoic acid (6)

Compound 5 (167 mg, 0.38 mmol) was incubated in concentrated ammoniumhydroxide (4.0 ml) for 1.0 hr at 50 degrees C. The resulting solutionwas evaporated to dryness and the residue was precipitated frommethanol-diethyl ether: 96-mg (74% yield). TLC (50/48/2, ethylacetate/methanol/acetic acid), R_(f)=0.50; ¹H NMR (DMSO-d₆) δ 7.78 (1H,m, aromatic-H), 7.43 (2H, m, aromatic-Hs), 7.18 (1H, m, aromatic-H),6.95 and 6.26 (2H, 2×s, aromatic-Hs), 2.37 (2H, t, J=6.6 Hz,methylene-Hs), 1.68 (2H, m, methylene-Hs). Note—An additional methylenepeak (2Hs) is expected to be hidden under the large water peak coveringthe range 3.8-3.1 ppm.

Reaction of 6 with Succinic Anhydride (Compound 7)

To a suspension of 6 (150 mg, 0.44 mmol) in a solution of anhydrous DMF(1.0 ml) and anhydrous triethylamine (0.34 ml) was added succinicanhydride (52 mg, 0.52 mmol). The reaction mixture was stirred for 18 hrat room temperature. The solvents were evaporated off and the residuewas purified by silica gel chromatography eluting with 50/50, ethylacetate/methanol. The UV-active column band was isolated and evaporatedaffording a homogeneous oil: 150 mg (77% yield).

BCSI-3 (Compound 1)

A mixture of 7 (150 mg, 0.34 mmol) and 7-chloro-1,6-dihydroxynapthalene(100 mg, 0.51 mmol), prepared as described in U.S. Patent ApplicationPublication No. US 2006/0204990, was stirred in a solution oftrifluoroacetic acid (1.4 ml)/methanesulfonic acid (0.50 ml) for 2 hr.at 50 degrees C., followed by continued stirring at room temperature for20 hr. The crude product was filtered and dried after precipitation byaddition of 6 ml of water to the reaction mixture. The crude product wasthen purified by silica gel chromatography eluting with 48/48/4,methanol/methylene chloride/triethylamine as the mobile phase. The pureproduct fractions were evaporated to dryness and the residue wassuspended in water. Sodium hydroxide solution (1 M) was added drop wiseto dissolve the material before addition of 1 M hydrochloric acidsolution to re-precipitated the pure dye product. The solid wasfiltered, rinsed with water and dried: 95 mg (49% yield). TLC (48/48/4,methylene chloride/methanol/triethylamine), R_(f)=0.56; ¹H NMR (DMSO-d₆)δ 8.04 (1H, dd, J=6.6 & 2.7 Hz, aromatic-H), 7.76 (3H, m, aromatic-Hs),7.40 (1H, d, J=9.0 Hz, aromatic-H), 7.35 (1H, s, aromatic-H), 7.26 (1H,dd, J=6.3 & 0.90 Hz, aromatic-H), 6.99 (1H, s, aromatic-H), 6.61 (1H, d,J=6.9 Hz, aromatic-H), 6.50 (1H, s, aromatic-H), 2.92 (2H, m,methylene-Hs), 2.50 (4H, m, methylene-Hs), 2.24 (2H, m, methylene-Hs),1.46 (2H, m, methylene-Hs).

The absorbance spectra of BCSI-3 are shown in FIGS. 24-26. FIGS. 24-26compare the absorbance spectra of EBIO-3 and BCSI-3 at pH 9.5, 7.4, and4.5 (borate buffer, phosphate buffered saline, and acetate buffer),respectively. The wavelength spectra are equivalent for the two dyes atall three pHs (high, mid, and low).

The purity (HPLC) was determined by UV/Vis absorption for BCSI-3was >99% at 530 nm and 95% at 270 nm.

The ¹H NMR spectrum of BCSI-3 was consistent with the dye's structure.

The pKa of BCSI-3 was determined by an absorbance assay with controlledpH buffers. The pKa results for BCSI-3 and EBIO-3 are shown in FIGS. 27and 28, respectively. The two pKa values are 6.64 (BCSI-3) and 6.61(EBIO-3).

A photobleaching degradation curve for EBIO-3 and BCSI-3 was constructedutilizing fluorescent light exposure and measuring the absorption at 530nm to quantify functional dye. Identical concentrations of both dyes,7.9 μM, were exposed to continuous fluorescent light (34 W Philips Hgbulb) from a distance of 16 inches for 3 days. The results are shown inFIG. 29. At 73.5 hours the two dyes had degraded 64% and the degradationis within 5.6% of each other. A control set of dyes protected from lightshowed unchanged absorptions at 530 nm.

Example 13 The Preparation and Fluorescent Properties of aRepresentative Fluorophore-Protein Conjugate BCSI-3/HSA

In this example the preparation and properties of a representativefluorophore-protein conjugate, BCSI-3/HSA, is described. The preparationis illustrated schematically in FIG. 30.

BCSI-3, prepared as described in Example 12, was conjugated torecombinant human serum albumin (rHSA) by an EDC(1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide hydrochloride)activation reaction. The dye was activated by a 1 hour reaction with EDCin an 83% DMF (N,N-dimethylformamide) and 17% PBS (phosphate bufferedsaline 150 mM sodium chloride, 50 mM sodium phosphate, pH 7.4) mixedsolvent. The EDC is at a 5 times molar excess to the dye.

The activated dye was mixed with a 13.6 mg/ml rHSA solution a pH 8.5carbonate buffer (100 mM sodium carbonate). The dye was at a 25 timesmolar excess to the protein. The conjugation reaction is incubatedovernight protected from light.

Purification of the conjugate from free dye was carried out byultrafiltration with PBS and a 10,000 MW cutoff (Amicon YM10,Millipore). The low molecular weight free dye was removed from thehigher molecular weight conjugate.

Conjugation by this method yielded a nominal 2 dye per rHSA conjugate.

EBIO-3 has a 3 carbon atom long carboxylic acid linker arm. This linkerlength allows an intramolecular ester to form with the adjacent phenolon the dye ring structure. This relatively stable 6-member lactone formsas an intermediate that likely protects the EBIO-3 dye from thehydrolysis during pH 8.5 reaction with lysine on the HSA. EDC activatedEBIO-3 routinely gives 2:1 loading level of dye:HSA from an offeringratio of 5:1. In contrast the BCSI-3 dye has an 8 atom long linker armthat cannot form the stable 6-member lactone ring structure. More rapidhydrolysis of the EDC activated BCSI-3 dye at pH 8.5 leads to lowerreaction efficiency in the conjugation reaction. EDC activated BCSI-3routinely gave 2:1 loading level of dye:HSA from an offering of 25:1.The excess dye was easily removed from the labeled protein by sizeexclusion chromatography or stirred cell. More amide bond formationoccurred with EBIO-3 than with BCSI-3. More EDC ester hydrolysisoccurred with BCSI-3 than with EBIO-3. Therefore more offered dye wasneeded for BCSI-3 to obtain similar loading.

FIG. 31 is a graph illustrating loading ratio of a representativefluorescent species (BCSI-3) to a representative protein (rHSA) as afunction of offering ratio (BCSI-3/rHSA). FIG. 32 compares fluorescentratio signal as a function of test buffer pH for representativefluorophore-protein conjugates (EBIO-3/HSA and BCSI-3/HSA conjugates)useful in the method and system of the invention.

Example 14 The Preparation and Fluorescent Properties of RepresentativeMembrane-Immobilized Fluorophore-Protein ConjugatesBCSI-3/HSA/Nitrocellulose

In this example, the preparation of representative membrane-immobilizedfluorophore-protein conjugate, BCSI-3/HSA/nitrocellulose, is described.

BCSI-3:rHSA conjugate, prepared as described in Example 13, wasimmobilized to a mixed cellulose acetate/cellulose nitrate filter paperof 0.22 um pore size (GSTF, Millipore). The protein conjugate wasdiluted to a 0.2 mg/ml solution in PBS pH 7.4 and added to the membraneat 70 ml per 100 cm². The immobilization reaction was incubated a roomtemperature with gentle agitation over night protected from light,washed three times with PBS, pH 7.4, two for 3 hours and one over night.The resulting paper was washed with a 1 hour CPS, pH 5.0 (50 mM citricacid, 100 mM sodium phosphate, 150 mM sodium chloride, pH 5.0) washfollowed by drying at room temperature protected from light.

The filter paper with immobilized conjugate is punched into ⅛ inch discsand assembled into the plastic pH sensors. The sensor consists of around cap with edges to hold the ⅛ inch disc in place and a barbed shaftwith a transparent window through which the fluorescent reading can bemade. When assembled the two plastic parts sandwich the membrane inplace against the window. The two parts are then ultrasonically weldedto each other.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method for measuring the pH of a sample, comprising: (a)irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe, wherein the fluorescent speciesimmobilized on the substrate is in liquid communication with a sample,wherein the excitation light has a wavelength sufficient to effectfluorescent emission from the fluorescent species, wherein thefluorescent species exhibits a first emission intensity at a firstemission wavelength and a second emission intensity at a second emissionwavelength, the ratio of the first and second emission intensities beingdependent on pH, and wherein the fluorescent species immobilized on thesubstrate is prepared by covalently coupling a benzo[c]xanthene linkerto the substrate, wherein the benzo[c]xanthene linker compound hasformulae (I) or (II)

their salts, active esters, acid/base forms, and tautomers, wherein atleast one of the X₀ groups is a halogen (e.g., chloro); R_(1′), R₁, R₂,R₃, and R₄ are each independently selected from hydrogen, halogen,cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and(C₁-C₈)alkoxy, aryl, and heteroaryl; X₁, X₂, X₃, and X₄ are eachindependently selected from the group consisting of hydrogen, halogen,cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl, aryl(C₁-C₄)alkyl, heteroaryl,SO₃H, and CO₂H; wherein the alkyl portions of any of R_(1′), R₁, R₂, R₃,and R₄, and X₁, X₂, X₃, and X₄ are optionally substituted with halogen,carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetylor hydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and wherein the aryl or heteroaryl portions of any ofR_(1′), R₁, R₂, R₃, and R₄, and X₁, X₂, X₃, and X₄ are optionallysubstituted with from one to four substituents selected from the groupconsisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;and wherein the linker arm has the formula—CH₂CH₂-L₁-NH-L₂-FG wherein L₁ has a length not exceeding the length ofa normal alkyl chain of 25 carbons and comprises from one to about 50atoms, wherein L₂ has a length not exceeding the length of a normalalkyl chain of 25 carbons and comprises from one to about 50 atoms, andwherein FG is a functional group reactive toward and capable ofcovalently coupling the fluorescent dye compound to a suitably reactivematerial; and (b) measuring the first and second emission intensities todetermined the pH of the sample.
 2. The method of claim 1, wherein thebenzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.
 3. The methodof claim 1, wherein the benzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.
 4. The methodof claim 1, wherein the substrate is a macromolecule.
 5. The method ofclaim 4, wherein the macromolecule is an albumin.
 6. The method of claim4, wherein the macromolecule is a human serum albumin.
 7. The method ofclaim 1, wherein the sample comprises blood or a blood product.
 8. Themethod of claim 1, wherein the probe is physically isolated from thefluorescent species immobilized on the substrate by a window transparentto the excitation light and the fluorescent emission.
 9. The method ofclaim 1, wherein the sample is contained within a sealed vessel.
 10. Asystem for measuring pH, comprising: (a) a light source for exciting afluorescent species immobilized on the substrate, wherein thefluorescent species has a first emission intensity at a first emissionwavelength and a second emission intensity at a second emissionwavelength, wherein the fluorescent species immobilized on the substrateis prepared by covalently coupling a benzo[c]xanthene linker to thesubstrate, wherein the benzo[c]xanthene linker compound has formulae (I)or (II)

their salts, active esters, acid/base forms, and tautomers, wherein atleast one of the X₀ groups is a halogen (e.g., chloro); R_(1′), R₁, R₂,R₃, and R₄ are each independently selected from hydrogen, halogen,cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkylthio and(C₁-C₈)alkoxy, aryl, and heteroaryl; X₁, X₂, X₃, and X₄ are eachindependently selected from the group consisting of hydrogen, halogen,cyano, trifluoromethyl, (C₁-C₈)alkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, aryl, aryl(C₁-C₄)alkyl, heteroaryl,SO₃H, and CO₂H; wherein the alkyl portions of any of R_(1′), R₁, R₂, R₃,and R₄, and X₁, X₂, X₃, and X₄ are optionally substituted with halogen,carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetylor hydroxy, and the alkyl portions of the substituents have from 1 to 6carbon atoms; and wherein the aryl or heteroaryl portions of any ofR_(1′), R₁, R₂, R₃, and R₄, and X₁, X₂, X₃, and X₄ are optionallysubstituted with from one to four substituents selected from the groupconsisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- ordi(C₁-C₆)alkylamino, (C₁-C₆)alkyl, (C₁-C₆)alkylthio and (C₁-C₆)alkoxy;and wherein the linker arm has the formula—CH₂CH₂-L₁-NH-L₂-FG wherein L₁ has a length not exceeding the length ofa normal alkyl chain of 25 carbons and comprises from one to about 50atoms, wherein L₂ has a length not exceeding the length of a normalalkyl chain of 25 carbons and comprises from one to about 50 atoms, andwherein FG is a functional group reactive toward and capable ofcovalently coupling the fluorescent dye compound to a suitably reactivematerial; (b) a first emission detector for measuring the first emissionintensity; (c) a second emission detector for measuring the secondemission intensity; (d) an excitation lightguide for transmittingexcitation light from the light source to the fluorescent species,wherein the lightguide comprises a first terminus proximate to the lightsource and a second terminus distal to the light source; (e) a firstemission lightguide for transmitting emission from the fluorescentspecies to the first emission detector, wherein the lightguide comprisesa first terminus proximate to the detector and a second terminus distalto the detector; (f) a second emission lightguide for transmittingemission from the fluorescent species to the second emission detector,wherein the lightguide comprises a first terminus proximate to thedetector and a second terminus distal to the detector; (g) a probehousing the distal termini of the excitation lightguide, first emissionlightguide, and second emission light guide; and (h) an assembly forreceiving the probe, the assembly comprising: (i) a housing forreceiving the probe, wherein the housing is adapted for receiving theprobe at a first end and terminating with a window at the second end,the window being transparent to the excitation and the emission light,(ii) a tip member reversibly connectable to the housing's second end,wherein the tip member is adapted to receive liquid from a sample to bemeasured, and (iii) the fluorescent species immobilized on the substrateintermediate the tip member and the window, wherein the fluorescentspecies immobilized on the substrate is in liquid communication with thesample during the measurement.
 11. The system of claim 10, wherein thebenzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.
 12. The systemof claim 10, wherein the benzo[c]xanthene linker compound is

its salts, active esters, acid/base forms, and tautomers.
 13. The systemof claim 10, wherein substrate is a macromolecule.
 14. The system ofclaim 13, wherein the macromolecule is an albumin.
 15. The system ofclaim 13, wherein the macromolecule is a human serum albumin.
 16. Thesystem of claim 10, wherein the probe is physically isolated from thefluorescent species immobilized on the substrate by a window transparentto the excitation light and the fluorescent emission.
 17. The system ofclaim 10, wherein the light source is a light-emitting diode.
 18. Thesystem of claim 10, wherein the first and second detectors arephotodiodes.
 19. The system of claim 10, wherein the sample comprisesblood or a blood product.
 20. The system of claim 10, wherein the sampleis contained within a sealed vessel.